Embryonic development and ‘physiological zero’

Embryonic development occurs in two stages, prior to the egg-laying – when the egg is still in formation in the oviduct – and after the egg-laying – when the egg is artificially incubated at 37.5°C and 60% humidity. Approximately three hours after the fertilisation in the infundibulum, embryonic development starts simultaneously with the formation of the egg in the oviduct; therefore, at the egg-laying time, embryos are either at the stage of pre-gastrula or in the initial stages of gastrulation (Barbosa, Reference BARBOSA2011).

The start of the embryo development takes place in the oviduct, where the hen's body temperature is 40 to 41°C. However, after the egg-laying occurs, the eggs experience a major change in external temperature. If this is below 24°C, then embryonic development will be interrupted. This is why the temperature of 24°C is considered to be the ‘physiological zero’ of the embryo. Accordingly, egg storage takes place at temperatures below the ‘physiological zero’ and embryonic development is resumed only when the eggs are incubated at the temperature of 37.5°C. Fasenko (Reference FASENKO2007) has challenged the ‘physiological zero’ definition, as the literature reports a wide variety of minimal temperatures for the embryonic development, from 21 up to 28ºC. Additionally, this author also states that, despite the embryonic development being not observed under the microscope, metabolic processes can occur below certain temperatures, so the ‘physiologic zero’ concept should be revised.

Room storage conditions

The storage temperature must be set according to the period required and the broiler breeders’ age. Eggs from young breeders are able to sustain longer storage periods under higher temperatures and lower humidity than those eggs from old broiler breeders. Egg storage for less than four days can at temperatures between 20 and 25°C. Where they are stored for four to seven days, the temperature must be approximately 16-17°C and, for eggs stored for more than seven days, temperature decrease to 10-12°C (Meijerhof, Reference MEIJERHOF1992).

In another study, Meijerhof (Reference MEIJERHOF1995) recommended temperatures between 18 and 20°C for storage periods of up to three to four days and application of high humidity (about 80%) in order to prevent excessive losses in egg weight. The author stressed that low temperatures can be applied to egg storage for short periods of time, thereby reducing the need for increased humidity in the storage room, as the loss of egg weight will be less at lower temperatures under similar relative humidities.

Reis et al. (Reference REIS, GAMA and SOARES1997), assessed time periods for storing eggs from young and old broiler breeders, and recommended the storage of only young breeders eggs whenever feasible. Brake (Reference BRAKE1999) recommended different conditions and storage periods for eggs from broiler breeders at distinct ages. Eggs from hens aged between 25 and 35 weeks should be stored at 50 to 60% humidity for up to four days, as, at such stage, the eggshell and albumen is thicker and highly resistant to degradation. Thus storage could be used to improve the incubation yield due to the albumen liquefaction which favours gas exchanges. Eggs from broiler breeders aged 36 to 50 weeks must be stored for two to five days at 18°C and 75% relative humidity. Concerning the storage of eggs from broiler breeders aged between 51 and 65 weeks, Brake recommended this should not exceed two to three days. Overall, whenever storage periods longer than four days are required, the room temperature must be reduced and the relative humidity must increase.

Ruiz and Lunam (Reference RUIZ and LUNAM2002) reported that storage temperatures of 16.5°C or 20°C did not affect the hatching of fertile eggs stored for short periods of time – one to three days. However for storage periods longer than nine days, a temperature of 10°C must be applied to reduce the baseline embryonic mortality and increase the hatching of fertile eggs.

Reijrink et al. (Reference REIJRINK, MEIJERHOF, KEMP and VAN DER BRAND2008) stated that cell death and metabolism still occur whenever eggs are stored at temperatures below, however near, the physiological zero. In order to reduce the embryonic development, eggs must be stored at temperatures of 10°C.

Change in the egg during storage

Eggs incubated immediately after laying show low incubation yields (Benton and Brake, 1996). The excellent quality of fresh egg albumen impairs the movement of nutrients from the albumen to the blastoderm, and gaseous exchange between the embryo and environment (Brake et al., Reference BRAKE, WALSH, BENTON, PETITTE, MEIJERHOF and PEÑALVA1997). Such effects are even more noticeable in eggs from young hens, which, as they are more efficient in the deposition of the egg white proteins, form a stable network and, consequently, a denser albumen. Additionally, the better quality of eggshell at this age makes the exchange of gases during incubation more difficult. Therefore, before being incubated, eggs are stored to increase albumen pH and to cause its liquefaction, as this leads to better hatching and embryonic growth rates.

Albumen pH is about 7.6 at the time of egg-laying and goes up to between 9.2 and 9.7 during storage. This is because a significant amount of bicarbonate and carbon dioxide is present in the egg albumen at the egg-laying time and, over time, there is a loss of CO₂ and increase in pH, leading to the denaturing of albumen proteins, which become more liquefied, reducing the Haugh Units (Lapão et al., Reference LAPÃO, GAMA and SOARES1999; Xavier et al., Reference XAVIER, CANÇADO, FIGUEIREDO, LARA, LANA, SOUZA and BAIÃO2008).

Whereas the albumen pH shows a rise in alkalinity in a short period of time, the yolk remains slightly acidic, with a pH of about 6.5, during storage (Lapão et al., Reference LAPÃO, GAMA and SOARES1999, Bakst and Holm, Reference BAKST and HOLM2003). Therefore, a concentration gradient of hydrogen ions 1000 times (three units of pH) can form between the blastoderm and its intermediary position between albumen and yolk. That means that the blastoderm faces a highly alkaline environment in the dorsal surface of the epiblast, while its ventral surface is in contact with a lightly acid environment. Such an position causes blastoderm pH to become intermediary and, according to Onagbesan et al. (Reference ONAGBESAN, BRUGGEMAN, DE SMIT, DEBONNE, WITTERS, TONA, EVERAERT and DECUYPERE2007), the optimum pH for gastrulation is of 8.2. However, the increase in albumen pH due to the storage period can influence blastoderm pH, thereby damaging initial embryo development.

Storage impacts the yolk index, a quality measure obtained from the division of the yolk height by its diameter. The index declines as a function of the length of the storage period because the vitelline membrane becomes weaker, thus reducing the yolk height and increasing its diameter (Kirunda and McKee, Reference KIRUNDA and McKEE2000). According to Reijrink et al. (Reference REIJRINK, MEIJERHOF, KEMP and VAN DER BRAND2008), albumen pH is the most important factor for vitelline membrane resistance and yolk index. Albumen pH affects the chalaziferous layer, which envelopes the yolk, and the vitelline membrane resistance is dependent upon this layer's viscosity. Since the increase in the albumen pH causes the albumen liquefaction, including the chalaziferous layer, the vitelline membrane resistance goes down and yolk index goes up. As the vitelline membrane and the chalaziferous layer protects the embryo during early incubation when the amnion is not yet completely formed, the disintegration of such structures during prolonged storage periods can negatively affect embryonic development.

Egg mass during storage and incubation

During storage and incubation, eggs lose weight due to the water loss that occurs via the diffusion processes through the eggshell pores. According to Tullett (Reference TULLETT1990), approximately 12% of the weight loss occurs within 18 days of incubation, and this gives the best hatching indexes. This weight loss depends on temperature, relative humidity, storage period, age and bird strain. The two last factors directly impact on the eggshell quality and thickness, which is negatively correlated to the egg weight loss (Roque and Soares, Reference ROQUE and SOARES1994).

Despite the weight loss during storage being small in comparison with that seen in the incubation period, Mayes and Takeballi (Reference MAYES and TAKEBALLI1984) recommend it must be minimised. Walsh et al. (Reference WALSH, RIZK and BRAKE1995) observed that 14-day-storage at temperature of 23.9°C caused excessive egg weight loss, impacting embryonic survival. These authors reported an oxygen consumption rate equivalent to the rate of water loss by the embryo during incubation. Therefore, if weight loss is excessive during storage, less water will be left during the incubation period, thus cutting down the input of oxygen to the embryo and causing initial embryonic mortality rates to rise.

Tona et al. (Reference TONA, BAMELIS, COUCKE, BRUGGEMAN and DECUYPERE2001) did not find any correlation between hatchability and embryonic mortality and egg weight-loss over the first 18 days of incubation, despite the fact they observed better hatching rates for eggs whose weight-loss was between 10.9 and 11.1% at 18 days incubation. These authors stated that excessive weight loss is detrimental because dehydration reduces the gas exchange during the embryonic development, in addition to damaging the hatching process itself. However eggs that lose little water can also harm hatching, because their embryos can ‘drown’ in the amniotic liquid. This is because, close to the hatching time, albumen remaining in the egg's thin tip enters the amniotic sac and forms a compound, which, due to the pressure difference resulting from the continuous embryonic growth, is orally consumed (Moran Jr., Reference MORAN2007).Whenever eggs lose little weight, there may be more albumen left and the ‘albumen-amniotic liquid’ compound will become more voluminous, and can cause the embryo to drown during its consumption.

Embryonic viability during storage

Meijerhof (Reference MEIJERHOF1992) reported that the reduction in hatchability due to storage is owing to the reduction in the embryo viability caused by changes occurring in the embryo and/or in the egg. Among these is the occurrence of cell necrosis, as demonstrated by Arora and Kosin (Reference ARORA and KOSIN1968), who found out an increased number of cells with necrotic nuclei with longer storage periods, and a reduction in embryo growth rates.

In a study conducted by Bloom et al. (Reference BLOOM, MUSCARELLA, LEE and RACHLINSKY1998), an increase in apoptotic cells in eggs stored for 14 days at 12°C and 50% relative humidity was observed when compared to fresh eggs (13.9% vs. 3.1%, respectively). According to such authors, these findings demonstrated that blastoderm cells are resistant to prolonged storage at low temperature; however they also suggested that a relatively modest quantity of cell death (14%) can affect embryo viability. According to Decuypere and Bruggeman (Reference DECUYPERE and BRUGGEMAN2007), egg storage for long periods can increase the number of necrotic cells or can predispose such cells to non-scheduled apoptosis. This causes an increase in the rate of non-viable embryonic cells, resulting in abnormal or dead embryos. The authors further stated that an optimal number of viable embryonic cells is necessary to initiate normal embryonic growth and development.

Published literature reports that fertile eggs can be stored for up to seven days with little or no effect on hatchability (Fasenko, Reference FASENKO2007). However, more than seven days causes embryonic abnormality and increased mortality, reducing the hatchability rate.

Storage, embryonic metabolism and incubation time

Visschedijk (Reference VISSCHEDIJK1968) demonstrated that the incubation period is determined by the amounts of O₂ and CO₂ present in the air chamber, therefore eggshell pipping will be accelerated if the O₂ levels are low and the amount of CO₂ is high, generally twice that of the oxygen content. Mather and Laughlin (Reference MATHER and LAUGHLIN1976; Reference MATHER and LAUGHLIN1977) attributed the increase in the incubation period of eggs stored for seven or 14 days, compared with incubated fresh eggs, to the delay in the start of embryonic development and to the lower rate of embryonic growth evidenced by the embryos in stored eggs. These authors explained that storage can delay embryogenesis, and the delay in embryonic development may be due to the lower rate of embryonic growth at the beginning of incubation.

According to Walsh et al. (Reference WALSH, RIZK and BRAKE1995), eggs stored for longer periods of time lose more weight through water evaporation during the storage and have less water available to be lost during incubation. Therefore, less oxygen will be available to the embryo during its development, thereby reducing cell metabolism and causing delay and reduction in the embryonic development.

Fasenko et al. (Reference FASENKO, ROBINSON, SEGURA, FEDDES and OUELLETTE2002) observed differences in the metabolism of embryos from eggs stored for four or 15 days. The embryos from eggs stored for four days showed larger production of gas within the period from four to 18 days of incubation and larger weight at 448 hours of incubation than those from eggs stored for 15 days.

Christensen et al. (Reference CHRISTENSEN, WINELAND, FASENKO and DONALDSON2002) observed an increase of 11 hours in the required incubation period whenever eggs had been stored for 14 days, in comparison with those stored for one day. According to Schmidt et al. (Reference SCHMIDT, FIGUEIREDO and AVILA2002), the activation of the early embryonic development occurs by pH-dependent enzymes. Prolonged storage excessively increases pH, negatively impacting on the onset of the embryonic development. Tona et al. (Reference TONA, MALHEIROS, BAMELIS, CAREGHI, MORAES, ONAGBESAN, DECUYPERE and BRUGGEMAN2003) evaluated the incubation period of eggs stored for three and 18 days and observed that hatching of eggs stored for shorter period took place between 478 and 494 hours of incubation, with a peak at 486 hours. However hatching of eggs stored for 18 days began only at 484 hours and the hatching peak was at 504 hours. These authors reported a longer period of incubation for eggs stored for 18 days, due to the delay in pipping of the eggshell inner membrane and because of the longer period required to complete eggshell pipping. This longer period was associated with the late increase in the production of corticosterone, which is required to stimulate the conversion of T₄ into T₃ during embryonic development. The hormone T₃ is negatively related to the concentration of pO₂/pCO₂, signalling its relationship with embryonic metabolic rate. These authors also found smaller amounts of CO₂ and higher amounts of O₂ in the air chambers of the eggs stored for 18 days, in comparison with the eggs stored for three days, both when evaluated at 18 days of incubation and at pipping. This indicates that the embryos of the eggs stored for 18 days were at an earlier embryonic stage than those stored for three days. According to Fasenko (Reference FASENKO2007), longer periods of incubation of eggs stored for longer times is due to slower development.

Storage impact on incubation yield

Lapão et al. (Reference LAPÃO, GAMA and SOARES1999) demonstrated that the hatching decline in the stored eggs begins one day after laying. In an epidemiologic study on the hatchability of broilers breeders eggs, Heier and Jarp (Reference HEIER and JARP2001) found out a 0.7% reduction in the hatchability per storage day. Elibol et al. (Reference ELIBOL, PEAK and BRAKE2002) evaluated the effect from three storage periods (3, 7 and 14 days) on incubation yield and found out a hatching decline in fertile eggs as the storage period increased.

Elibol and Brake (Reference ELIBOL and BRAKE2008) observed a reduction in hatching rate due to the increase in initial embryonic mortality (zero to six days of incubation) and late embryonic mortality (18 to 21 days of incubation, including pipped eggs), when the eggs were stored for 14 days compared to those stored for three days.

According to a field study, based on the three year data from three Dutch incubation chambers, Yassin et al. (Reference YASSIN, VELTHUIS, BOERJAN, VAN RIEL and HUIRNE2008) observed that, until the seventh storage day, each additional storage day reduced hatchability by 0.2%. From seven days on, such hatching reduction rises by 0.5% per extra storage day. In an assessment of the effects of egg storage for two, four, six, eight, ten, 12 and 14 days on embryonic mortality and hatchability, Schmidt et al. (Reference SCHMIDT, FIGUEIREDO, SAATKAMP and BOMM2009) observed the linear relationship between storage period and embryonic mortality and hatching. Each additional storage day of the eggs caused a reduction of 1.17 and 1.15% in the hatchability and embryonic mortality, respectively.

Tanure et al. (Reference TANURE, CAFÉ, LEANDRO, BAIÃO, STRINGHINI and GOMES2009) observed a reduction in the hatching rate of eggs from light breeders (aged 57 weeks old) which were stored for seven days in comparison with three and five days. When comparing the storage of eggs from old broiler breeders (59 wk) for three and seven days, Rocha (Reference ROCHA2011) observed a decline in hatchability due to increased embryonic mortality after 15 days of incubation.

Chick weight as a function of the fertile egg storage

Ruiz and Lunam (Reference RUIZ and LUNAM2002) observed that chicks from eggs stored for nine to 11 days, at 10ºC, were heavier at hatching than those from eggs stored for the same time period at higher temperatures. The authors did not explain their findings, however it may have been due to a smaller loss of egg weight during storage at lower temperatures, resulting in larger amounts of water to be evaporated during incubation, assuring greater provision of oxygen to the embryo and, therefore, greater metabolism and deposition of tissue.

Short storage periods can harm the chick weight. In an assessment of the weight of chicks from eggs stored for two, four, six and eight days, Ferreira (Reference FERREIRA2006) found lower body weights in the chicks hatched from the eggs stored for two days, in comparison with the other storage periods. The author credited this inferior embryonic development to the lower liquefaction of the albumen during the short storage, resulting in lower provision of oxygen and nutrients to the embryos and lower deposition of body tissue. Similarly, Tanure et al. (Reference TANURE, CAFÉ, LEANDRO, BAIÃO, STRINGHINI and GOMES2009) observed that egg storage for three days resulted in lighter chicks at the hatching time, in comparison with eggs stored for seven days.

Handling during storage

Placing the eggs with the fine tip upwards is the recommended management for eggs stored for long periods (Antwi, Reference ANTWI1993). When the egg is stored for long period with the large end up, the albumen degrades and the yolk rotates and floats to the top of the egg, positioning the blastoderm near to the shell. This facilitates additional dehydration of the embryo and exposure to bacterial contamination, resulting in a greater risk of embryonic mortality (Adda, Reference ADDA2003). Elibol and Brake (Reference ELIBOL and BRAKE2008) observed better hatching of eggs stored with the fine edge upward at 14 days compared to the hatching of eggs stored for the same period, but with the round end up. This effect of the position of the egg was not observed when eggs were stored for three days. The improvement in hatching for eggs with fine edge positioned upwards during prolonged storage may be associated with maintenance of the yolk centre position until incubation, ensuring the equatorial location of the blastoderm, thus maintaining conditions similar to that observed during laying.

According to Schmidt et al. (Reference SCHMIDT, FIGUEIREDO and AVILA2002), eggs stored for short periods (two to four days) do not require special handling, but for periods longer than four days it is necessary to use more specialised techniques. The use of a turning system in the storage room, similar to the egg turning in the incubator, is another practice that allows improvements in hatchability. However, Elibol et al. (Reference ELIBOL, PEAK and BRAKE2002) carried out two experiments with eggs from young broiler breeders (30 and 31 weeks old) and old (52 and 53 weeks) stored for three, seven or 14 days and subjected to zero, four or 24 turns per day during storage. The authors found that, regardless of the storage period, the turning of the eggs at least four times daily during storage improved hatchability.

Mayes and Takeballi (Reference MAYES and TAKEBALLI1984) have reviewed other management recommendations of eggs during prolonged storage, such as packaging for eggs in permeable plastics, with or without addition of gas (carbon dioxide and nitrogen) to avoid changes occurring in egg components, especially in the pH of the albumen.

Heating eggs before storage

Embryos at the pre-gastrula stage after laying are less resistant to storage stress than the embryos at gastrula stage (Butler, Reference BUTLER1991). The effect of this disadvantage can be reduced by heating the eggs shortly after laying, as demonstrated by Fasenko et al. (Reference FASENKO, ROBINSON, WHELAN, KREMENIUK and WALKER2001). The authors incubated eggs at 37.5°C for zero, six, 12 and 18 hours, and then stored the eggs for four and 14 days at 11.5°C and 60.5% relative humidity. There was no effect of pre-storage incubation on hatching in eggs stored for four days. However incubation for six hours before egg storage for 14 days improved hatchability by 8.5% when compared to non-incubated eggs (79.0% vs. 70.5%). Incubation for 18 hours before egg storage for 14 days increased early embryonic mortality, reducing hatching rate to 9.1%. The authors found that pre-storage incubation for six hours allowed embryos to complete the hypoblast formation, which is the initial stage of gastrulation, ensuring their survival during prolonged storage. In contrast, incubation for 18 hours before egg storage for 14 days advanced embryonic development to complete formation of the primitive streak. This advanced stage of gastrulation is characterised by active migration and differentiation, and the embryo is highly sensitive to changes in temperature over long periods.

Lourens (Reference LOURENS2006) confirmed this positive effect of pre-storage incubation with broiler breeder eggs hatchability. The author found that the incubation of eggs at 37.8°C and 55% relative humidity for three and six hours before storage for 14 days improved the fertile hatchability rate when compared to the control group which was not incubated before storage for the same period. Further incubation for three hours before storing eggs for a week also showed positive effects on hatchability.

Although Fiúza et al. (Reference FIÚZA, LARA, AGUILAR, RIBEIRO and BAIÃO2006) did not assess long egg storage periods, they kept eggs at 30°C after collection for zero, five and ten hours before storage for four days at 20°C. The authors observed that eggs held at a temperature of 30°C for five hours after the collection had better incubation yield. At this temperature, embryonic development increased to a stage that was more resistant to storage damage. The storage immediately after egg collection increased early embryonic mortality and reduced hatchability, probably due to the higher number of embryos in a pre-gastrula stage which would be more sensitive to cool temperatures.