138 Acta argiculturae Slovenica, Supplement 5, 138–142, Ljubljana 2016 24th Int. Symp. “Animal Science Days”, Ptuj, Slovenia, Sept. 21st−23rd, 2016. COBISS: 1.08 Agris category code: L01 EFFECTS OF FLOCK AGE, PRESTORAGE HEATING OF EGGS, EGG POSITION DURING STORAGE AND STORAGE DURATION ON HATCHABILITY PARAMETERS IN LAYER PARENT STOCK Dušan TERČIČ 1, Mojca PESTOTNIK 1 Effects of flock age, prestorage heating of eggs, egg position during storage and storage duration on hatchability parameters in layer parent stock 1 University of Ljubljana, Biotechnical Faculty, Department of Animal Science, Groblje 3, SI-1230 Domžale, Slovenia ABSTRACT This study was carried out to determine the effect of different preincubation storage periods, different prestorage egg treatments and different egg positions during storage on egg weight loss, embryo mortality, hatchability, hatchling weight and chick sex ratio at hatch in layer parent stock. The following factors were analyzed: flock age (24 weeks, 65 weeks), prestorage heating periods (0, 5 hours at 37.8 °C), storage periods (0, 3, 6, 9, 12, 15 days at 15.0 °C) and egg positions (pointed end up, pointed end down). Parent stock age had a significant (p < 0.05) effect on chick weight. This increase in chick size due to advancing age of layer parent stock is attributable to the fact that egg weight significantly (p < 0.05) improved as the age of parent stock increased. Significant differences were found in absolute egg weight loss during the storage period, as a function of flock’s age (p < 0.05). Eggs stored with the small end down lost less weight during storage compared with the eggs stored with the small end up. Hatchability of fertile eggs did significantly decline after storage for 9, 12, and 15 days. Flock age, prestorage heating and egg position during storage had no positive or det- rimental effect on hatchability of set and fertile eggs and to male:female ratio under the conditions of this study. A flock age x egg position interaction indicated that females, hatched from eggs derived from old flock exhibited a tendency to be heavier when eggs are stored pointed end down whereas the egg position did not affect the female weight when eggs came from young flock. Key words: poultry, layer parent stock, hatchability, prestorage heating, egg position, flock age 1 INTRODUCTION In commercial poultry production, storage of hatch- ing eggs is an indispensable part of hatchery operation and long egg storage times are sometimes unavoidable due to flock logistic issues and market availability (Lima et al., 2012). One of the most frequently used methods to reduce the negative effects of long-term storage has been to heat eggs for short periods before storage. Pre-storage heating is based on the fact that embryos at the pre-gas- trula stage at oviposition are less able to withstand the stress of storage compared with embryos at the later, gas- trula stage (Schulte-Drüggelte, 2011). In the recent dec- ades, several studies reported that pre-heating of poultry eggs before storage resulted in more live chicks and a lower level of embryonic mortality compared to the eggs that remained un-heated (Petek and Dikmen, 2004; Rei- jrink et al., 2009). The traditional way to store hatching eggs is in the large end up position. It is assumed that in the case of large end up position the embryo has a higher chance to dehydrate or to stick to the internal eggshell membrane than when the egg is stored in the pointed end up position (Reijrink, 2016). Beside length of stor- age, embryo developmental stage at the start of storage, the position of eggs during storage there are several other aspects of egg storage that can influence subsequent suc- cessful incubation: flock age, genetics, environmental conditions and egg chemical and physical characteristics (Schmidt et al., 2002). Some of these factors present in- teractions, and the work reported here was carried out with the aim to obtain further information on this sub- ject through the use of layer parent stock’s eggs subjected Acta agriculturae Slovenica, Supplement 5 – 2016 139 EFFECTS OF FLOCK AGE, PRESTORAGE HEATING OF EGGS ... HATCHABILITY PARAMETERS IN LAYER PARENT STOCK to different treatments prior to incubation. Most studies associated with the effect of egg storage period on hatch- ability characteristics are focused on Japanese quail and broiler parent stock. However, these parameters, espe- cially sex ratio at hatch have not been fully examined in layer parent stock. 2 MATERIAL AND METHODS In total, 4,349 freshly laid eggs (unwashed, feces- free) were obtained from two flocks of Slovenian layer parent stock Prelux-G. The parent stocks were housed in floor housing system. One flock consisted of young par- ent stock (24 weeks of age at the start of egg collection), and the other of old parent stock (65 weeks of age at the start of egg collection). In order that all the eggs from all groups could be set in the incubator at the same time, eggs were collected in time intervals, representing the six storage periods (0, 3, 6, 9, 12 and 15 days). Therefore, the first collection corresponded to eggs stored for 15 days and the sixth to fresh eggs (0 days). At the day of collec- tion all the eggs belonging to the particular flock were randomly distributed into the following four treatments: (1) prestorage heating and further storage in egg cooling room with the pointed end up; (2) prestorage heating and further storage in egg cooling room with the pointed end down; (3) holding eggs in egg cooling room with pointed end up; and (4) holding eggs in egg cooling room with pointed end down. A total of 48 interactive groups con- stituted this study: 2 flocks (young-old) x 6 storage peri- ods x 2 systems of storage treatments (prestorage heating: yes-no) x 2 positions of the hatching eggs during storage period (pointed end up – pointed end down). Prestorage heating was practiced in a single stage setter at a standard incubation temperature of 37.8 °C and 60 % RH on day 1 of storage and lasted 5 hours. Eggs were preheated and in- cubated in a Petersime model S168 setter and a Petersime model H168 hatcher (Petersime, Zulte, Belgium). After the prestorage heating treatments were completed, all the eggs including eggs from control and prestorage heating groups were stored in egg cooling room at a temperature of 15 °C and relative humidity of 75 % for 3, 6, 9, 12 or 15 days. After storage, eggs were transported from the cooling room to a commercial hatchery where they were in order to prevent condensation of moisture from the air on egg shell surface maintained at 22.0 °C and 60 % air relative humidity for three hours before incubation. Thereafter they were marked, weighed, set on the setter trays with pointed end down and disinfected by formal- dehyde fumigation for 20 min in the incubator before the incubation was initiated. For the first 18 days of incuba- tion, the setter temperature was 37.8 °C and humidity 60 %. For the rest period of 3 days the temperature in the hatcher was 37.2 °C and the humidity was 75 %. To de- termine weight loss eggs were weighed before incubation and at the moment of transference from setter to hatcher. Each setter tray contained eggs of 1 replication (out of 3) and therefore, the setter tray was used as the smallest experimental unit. Hatching was completed on the 21st day. The chicks were carefully removed from the duly identified hatching boxes according to treatment group, feather sexed and their weights were determined by us- ing an electronic scale. Eggs which did not hatch were broken onto a tray and identified for infertility or em- bryonic mortality. Fertility was calculated as a percentage of set eggs. Hatchability was calculated as a percentage of set eggs and as a percentage of fertile eggs. Embryon- ic mortality was calculated as a percentage of set eggs. Data were submitted to analysis of variance using the the PROC GLM procedure of SAS software (Sas Inst., 2011). To obtain a normal distribution all the data in percentage form were transformed using arc-sine transformations prior to analysis. The Tukey test was applied to detect sta- tistically significant (p < 0.05) differences between treat- ment groups. 3 RESULTS AND DISCUSSION As it is indicated in Table 1 egg weight at set was significantly (p < 0.05) affected by layer parent stock’s age. The eggs from old flock were (p < 0.05) larger than those from young flock. It is well recognized that there is a strong relationship between parent stock age and egg weight such that older hens produce larger eggs (Lourens et al., 2006). Significant differences were found in abso- lute egg weight loss during the storage period, as a func- tion of flock’s age (p < 0.05). Data showed that egg weight losses expressed in grams increased with advancing the age of parent stock hens (Table 1). Large eggs have a greater proportion of albumen (and thus greater moisture content) than small eggs. On the other hand eggs from older hens generally have a higher number of pores per egg and a very poor degree of cuticle coverage (Navarro et al., 2013). The cu- ticle somewhat seals the pores and is useful in reducing moisture losses. Egg weight loss expressed in percentages did not differ between two flocks. Age of the hens had a significant (p < 0.05) effect on chick weight (Table 1). Data revealed that male and female chick weights were amplified (p < 0.05) with increasing the flock age and consequently egg size. Since chick weight increased with advancing age of layer parent stock it is advisable to set eggs from older flocks in order to obtain havier chicks. Lower hatchability and higher percentage of dead em- Acta agriculturae Slovenica, Supplement 5 – 2016140 D. TERČIČ and M. PESTOTNIK bryos were recorded in older hens at the age of 65 weeks, however, non-significant difference (p > 0.05) was ob- served between these parameters at the age of 24- and 65-weeks-old hens (Table 1). In general, the decline in hatchability of fertile eggs with increasing age of the par- ent stock is caused by embryonic mortality. However many other contributing factors exist including larger egg size, poorer shell quality due to bigger surface area, albumen quality deterioration and increased the yolk cholesterol content (Iqbal et al., 2016). Egg weight at set was significantly (p < 0.05) reduced when eggs were preheated for 5 hours before storage (Table 1). These re- sults were expected because an exposure to heat treat- ment would increase the opportunity for water vapour to escape from the egg. Although prestorage heating treat- ment increased egg weight loss compared with the not preheated group, the differences in egg weight loss dur- ing storage did not affect the female or male weight, em- bryonic mortality, infertility and hatchability (Table 1). Some researchers claimed that warming of eggs did not affect hatchability when the eggs were stored for 4 to 7 days (Reijrink et al., 2009) or had even detrimental ef- fect on the hatchability when eggs were stored for 15 days (Petek and Dikmen, 2006). These differences might be due to variations in the stage of embryonic development at egg collection (before prestorage heating), genotype, flock age, egg yield, hatching performance, storage pe- riod and conditions, time of warming, as stated by other researchers (Elibol, 1997). The results presented in Ta- ble 2 gave a strong indication that egg position during storage has an influence (p < 0.05) on egg weight loss; positioning hatching eggs with pointed end up caused an increase in egg weight loss. It was suggested that storing eggs small end up may result in the central placement of the yolk in the albumen, giving the embryo greater protection from dehydration and adhesions (Mayers and Takeballi, 1984). These results do not agree with the re- sults of our study since storing the eggs pointed end up just insignificantly improved hatch of eggs set by 2.53 % and hatch of fertile eggs by 2.23 % which was probably due to less embryonic mortality (Table 2). No differences were found in the eggs’ fertil- ity among the different storage groups (p > 0.05) (Ta- ble 2). Hatchability of fertile eggs decreased significantly (p < 0.05) with increasing egg storage period after the 6th day of storage. The former results are consistent with the findings of (Elibol et al., 2002), who reported that hatchability after 7 days of storage was higher than that of eggs stored for 14 days in broiler hatching eggs. Stor- age for 6 days elevates the hatchability of fertile eggs over the fresh egg (Table 2). As an egg ages, it loses CO2 and moisture through shell pores, the albumen and yolk pH increase, the albumen height and vitelline strength de- crease. The egg must lose some of the CO2 to improve its hatchability but the loss must come at the right time. Excess carbon dioxide loss leading to a high albumen pH may have a negative effect upon initiation of embryonic growth and viability (Onagbesan et al., 2007). This situ- ation may explain why the eggs incubated the same day as they were laid did not hatch as well as those stored for period of 6 days (Table 2). Overall embryo mortal- ity went from 8.44 % in embryos of 6-days stored eggs to 16.86 % in embryos from 9-days stored eggs and this difference was found to be significant due to the main effect of egg storage duration (p < 0.05) (Table 2). When eggs age, the vitelline membrane which is an important Hatchability parameters Parent flock age Egg storage treatment Young flock (LSM ± SE) Old flock (LSM ± SE) Prestorage heating (LSM ± SE) Without prestorage heating (LSM ± SE) Egg weight at set (g) 56.77a ± 0.07 66.63b ± 0.13 61.46a ± 0.09 61.94b ± 0.09 Egg weight loss (g) 6.32a ± 0.03 7.49b ± 0.05 6.97a ± 0.04 6.85b ± 0.04 Egg weight loss (%) 11.14a ± 0.05 11.26a ± 0.09 11.34a ± 0.07 11.05b ± 0.07 Female weight (g) 46.82a ± 0.27 53.28b ± 0.48 49.65a ± 0.36 50.45a ± 0.36 Male weight (g) 47.43a ± 0.26 53.33b ± 0.45 50.09a ± 0.34 50.68a ± 0.34 Dead embryos (%) 11.36a ± 0.010 13.52a ± 0.030 12.61a ± 0.018 12.24a ± 0.018 Infertile eggs (%) 9.37a ± 0.007 9.05a ± 0.022 9.01a ± 0.013 9.42a ± 0.013 Hatchability of eggs set (%) 78.46a ± 0.007 76.10a ± 0.021 77.40a ± 0.012 77.19a ± 0.012 Hatchability of fertile eggs (%) 87.27a ± 0.010 85.52a ± 0.031 86.22a ± 0.018 86.60a ± 0.018 Male:female ratio 1.09a ± 0.041 1.16a ± 0.072 1.09a ± 0.058 1.16a ± 0.058 Table 1: The effect of parent flock age and egg storage treatment prior incubation on hatchability parameters LSM – least square mean; SE – standard error; a, b – means within the same row with different superscripts are significantly (p < 0.05) different Acta agriculturae Slovenica, Supplement 5 – 2016 141 EFFECTS OF FLOCK AGE, PRESTORAGE HEATING OF EGGS ... HATCHABILITY PARAMETERS IN LAYER PARENT STOCK barrier to prevent bacteria transfer between the albumen and yolk weakens (Gast et al., 2005; Kirunda and McKee, 2000). This weakening of the vitelline membrane, expos- es the embryo to levels of high alkaline pH, which it is suggested, is responsible for early embryonic mortality (Reijrink et al., 2008). Egg weights at set remained fairly constant till the 9th day of storage (Table 2). After that day egg weight significantly (p < 0.05) decreased. Statis- tical differences were detected for egg weight loss when eggs were stored for 15 days. A number of environmental factors have been suggested to influence the sex of the avian offspring produced (Pike and Petrie, 2003). Due to financial implications and animal welfare concerns skew- ing the sex ratio at hatch simply by prestorage treatment would be beneficial to the commercial poultry industry. Because only the hen is productive for the egg industry, considerable value would be added if the sex ratio of egg laying breeds was altered towards production of females. Our study did not find any skew in chicken sex ratios at hatch due to preincubation treatments. Males were more common than females but differences were insignificant (p > 0.05). These results are not in agreement with the findings of Boerjan (2016) who reported that the intro- duction of 3–6 hours’ prestorage heating in Lohmann layer hen hatcheries produced 3–7 % more females from eggs stored for more than 11 days. Interestingly, there was a significant interaction of flock age with egg position for female weight. The interaction was due to female weight decreased (2.4 g) with setting hatching egg with pointed end up in the old flock but not in the young flock. Also consistent with this result, there was a significant flock age x egg position interaction observed for egg weight loss due to higher egg loss for eggs set pointed end up from old flock (0.45 g; 0.73 %) but not from young flock. 4 CONCLUSIONS AND RECOMMENDA- TIONS a. Prestorage heating of hatching eggs for 5 hours at 37,8 °C was not an effective way to reduce the loss of hatchability in eggs stored for long periods. b. Storing eggs pointed end up significantly in- creased egg weight loss and brought some insignificant Hatchability parameters Egg storage duration Egg position during storage 0 d (LSM ± SE) 3 d (LSM ± SE) 6 d (LSM ± SE) 9 d (LSM ± SE) 12 d (LSM ± SE) 15 d (LSM ± SE) Pointed end down (LSM ± SE) Pointed end up (LSM ± SE) Egg weight at set (g) 62.37a ± 0.18 61.79ab ± 0.18 61.81ab ± 0.18 62.22a ± 0.18 61.22bc ± 0.18 60.77c ± 0.18 61.79a ± 0.10 61.61a ± 0.10 Egg weight loss (g) 6.86ab ± 0.08 6.73b ± 0.08 7.04ab ± 0.08 7.17a ± 0.08 6.81b ± 0.08 6.84ab ± 0.08 6.81a ± 0.04 7.01b ± 0.04 Egg weight loss (%) 11.02ab ± 0.13 10.92a ± 0.13 11.39ab ± 0.13 11.51b ± 0.13 11.12ab ± 0.13 11.23ab ± 0.13 11.03a ± 0.07 11.37b ± 0.07 Female weight (g) 49.13ab ± 0.68 49.01ab ± 0.68 48.53a ± 0.68 49.96abc ± 0.68 51.57bc ± 0.68 52.10c ± 0.68 50.57a ± 0.39 49.53a ± 0.39 Male weight (g) 50.69ab ± 0.63 48.44a ± 0.63 48.82a ± 0.63 52.36b ± 0.63 50.26ab ± 0.63 51.72b ± 0.63 50.53a ± 0.36 50.23a ± 0.36 Dead embryos (%) 10.40abc ± 0.060 9.70ab ± 0.060 8.44a ± 0.060 16.86c ± 0.060 14.16abc ± 0.060 16.08bc ± 0.060 13.57a ± 0.020 11.32a ± 0.020 Infertile eggs (%) 8.36a ± 0.044 7.95a ± 0.044 9.13a ± 0.044 7.73a ± 0.044 10.39a ± 0.044 12.05a ± 0.044 9.48a ± 0.015 8.95a ± 0.015 Hatchability of eggs set (%) 80.57ab ± 0.043 81.62a ± 0.043 81.32ab ± 0.043 74.27bc ± 0.043 74.35bc ± 0.043 70.85c ± 0.043 76.02a ± 0.014 78.55a ± 0.014 Hatchability of fertile eggs (%) 88.44abc ± 0.063 89.41ab ± 0.063 91.74a ± 0.063 81.57c ± 0.063 84.13bc ± 0.063 81.72c ± 0,063 85.28a ± 0.021 87.51a ± 0.021 Male : female ratio 1.04a ± 0.101 1.21a ± 0.101 1.06a ± 0.101 0.96a ± 0.101 1.23a ± 0.101 1.23a ± 0.101 1.14a ± 0.058 1.10a ± 0.058 Table 2: Effects of storage time and of egg position during storage on hatchability parameters LSM – least square mean; SE – standard error; a, b – means within the same row with different superscripts are significantly (p < 0.05) different Acta agriculturae Slovenica, Supplement 5 – 2016142 D. 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