Hypercapnia or hypoxia can cause changes in the physiology of embryos with respect to the control and timing of the hatching events The physiological changes can be induced in the pulmonary and circulatory system by chronic hypercapnia. This observation has resulted in the view that higher levels of CO₂ can shorten the effects of hypoxic conditions on developing embryos. Increasing CO₂ early or at the end of incubation acts as a hatching stimulus but also the hypoxic condition of high-altitude incubation also affects hatching events as well as hormonal levels. In fact, Hassanzadeh et al. (2004) showed that embryos incubated at high altitude had higher plasma triiodothyronine (T₃), thyroxin (T₄), and corticosterone levels and hatched earlier than those incubated at low altitude. El-Hanoun et al. (2019) reported that duck breeders’ eggs incubated under hypercapnic conditions hatched earlier than those incubated under normal conditions, and the hatch window was narrower. The authors demonstrated that this phenomenon is strongly related to increased levels of corticosterone, T₃ and T₄ as a result of increased pCO₂ (De Smit et al., 2006) in the air cell at internal pipping in hypercapnic condition (Figure 2). Thus the positive effects of hypercapnic incubation suggest an increase of T₃ and air cell pCO₂ resulting in the early hatch and enhanced hatchability.

The study of Tona et al. (2004) on non-ventilation during early incubation in combination with dexamethasone administration at two stages of development (d16 or d18) in embryos has elucidated the importance of timing in manipulating the hypothalamic-pituitary-adrenal (HPA) axis. Indeed, the authors reported that dexamethasone injected on day 18 raised the plasma T₃ levels (Table 1) at internal pipping (IP) and advanced hatching and reduced the hatching process. However, injection on day 16 had no effect on the plasma T₃ levels at IP. Also, dexamethasone injection on day 16 resulted in a rebound effect on the functioning of the HPA axis in early postnatal life, which was not observed in chickens injected on day 18. This disturbance in HPA axis establishment may cause an increased functioning and has been reviewed earlier (Decuypere and Michels, 1992).

Moreover, exposure of embryos to low O₂ or higher CO₂ resulted in significantly higher haematological parameters (Hb, PCV %, and RBC counts). Increased Hb under hypercapnia or hypoxia conditions is known to raise the oxygen-carrying capacity of the blood and represent an adaptive physiological response. The findings of Mortola (2004) indicated a stimulatory role of CO₂ on the chemoreceptors that enhance breathing efficiency and that hyperoxia at this period decreased the effect of hypercapnia. Hence, hypercapnia can achieve a similar effect as hypoxia on lung function during internal and external pipping and hatching.

These findings indicate that embryos adapted to hypoxic or hypercapnic conditions by enhancing angiogenesis processes, which subsequently increases their blood oxygen-carrying capacity, which positively affects their growth development and maturation. Such alterations may induce permanent phenotypic changes in the embryo, which may have a long term epigenetic effect on post-hatch performance.

Joseph et al. (2006) highlighted that a continuous low eggshell temperature of 36.6°C during the first 10 days of incubation reduced embryonic weight, hatchability and chick quality. Nideou et al. (2019) observed an increase in albumen utilization and growth of Isa brown embryos subjected to elevated temperature (38.5°C) during the first 10 days of incubation while the incubation time was shortened. According to Maatjens et al. (2017), a negative effect of an eggshell temperature of 38.9°C was observed from E15 onward and emphasize that an eggshell temperature of 35.6 and 36.7°C from E15 onward might be beneficial for chick embryo physiology. However, Yildirim and Yetisir (2004) studied the effect of different hatcher temperatures (36.1; 37.2; 38.3, and 39.4°C from 17 days of incubation until hatch with relative humidity at around 75% in all groups) on hatching traits. They found that the control group (37.2°C) had a better hatchability compared to the low-temperature group (36.1°C) which had low metabolic activity and then a high late embryo mortality rate. Wilson (1991) reported that in the last third of the incubation phase, a decrease in the temperature of incubation did not have a significant effect on hatchability but elongated incubation duration and decreased water loss. According to Tzschentke and Hall (2009), a temperature of 36.2°C from day 18–21 of incubation improved hatchability. Concerning the effect of a high temperature in the hatcher; Yildirim and Yetisir (2004) found that a temperature of 38.3°C in the hatcher allows for a hatchability similar to the control group (37.2), but a very high temperature (39.4°C) negatively affect hatchability by increasing late embryo mortality. Their results are not similar to those of Joseph et al. (2006) who found a better hatchability with eggs subjected to a higher hatcher temperature (39.5°C) compared to the control group (37.8°C).

Joseph et al. (2006) found that a continuous low eggshell temperature of 36.6°C during the first 10 days of incubation reduced live weight at 6 weeks of age and carcass yields. But increasing the incubation temperature (38.5°C) during the first 10 days of incubation did not impact the T₃ concentration of the hatched chicks and their post-hatch performance. Maatjens et al. (2016) showed that a high eggshell temperature (38.9°C) applied from day 15 of incubation on Ross eggs negatively impacted chick’s growth and FCR during the first week of rearing, while this post-hatch performance was improved compared to control batches when eggs were subjected to a temperature of 36.7°C from day 15 of incubation. According to İpek and Sözcü (2015), the carcass weight and yield are negatively affected by higher hatcher temperature but slaughter weight at higher hatcher temperature was similar to the control group. Joseph et al. (2006) stated in their study that high eggshell temperature in the hatcher reduced bodyweight and one-week weight gain. However, by three weeks of age, there was no difference in body weight between chicks in high eggshell temperature and control eggshell temperature treatments.

Increasing incubation temperature at 39.5°C during 24 h/day from E7 to E16 reduced hatchability by 25% and negatively affected the quality and the bodyweight of the hatched chicks while a thermal treatment at 65% during 12 h/day from E7 to E16 did not affect hatchability and bodyweight of cobbs hatched chicks (Piestun et al., 2008a). Tzschentke and Hall (2009) revealed that neither short-term (38.2°C–38.4°C, 2 h daily) nor chronic (38.2°C–38.4°C, 24 h daily) increase in incubation temperature in the hatcher (during the last four days of incubation) adversely affected hatchability and chick quality in broiler chickens. The body temperature was significantly reduced in broiler chicks subjected to thermal treatment (12 and 24 h/day) compared to the control group, but those subjected to a thermal treatment during 24 h/day had a body temperature lower than those of 12 h/day (Piestun et al., 2008a). This aligns with the reduced plasma triiodothyronine and thyroxine of the birds of 24 and 12 H groups and the plasma corticosterone increased with time.

Increasing incubation temperature at 39.5°C during 12 h/day from E7 to E16 improved the feed conversion ratio of broilers compared to the control (Piestun et al., 2013; Meteyake et al., 2020) without affecting broilers' growth compared to 24 h/day group which had lower body weights (Piestun et al., 2008b). According to Tzschentke and Hall (2009), the FCR of short-term warm stimulated broilers (38.2–38.4°C, 2 h per day, from d17 onward) was significantly lower than in broilers of the control (37.2–37.4°C) and chronic warm (38.2–38.4°C, 24 h per day, from d17 onward) incubated groups. The daily feed intake and weight gain were significantly lower in the short-term warm stimulated ducks than those of the control group in the first three weeks while short-term cold stimulation improved feed conversion ratio during the whole growing period exclusively in male ducks (Halle et al., 2012). According to Sgavioli et al. (2016), incubating eggs at 39°C compromises the body and heart development of layer chicks and reduces the availability of blood ionized calcium for bone mineralization during embryo development. Morita et al. (2016) concluded in their study that changes in chicken stickiness and vascularity as well as changes in thyroid and growth hormone levels are the results of embryonic strategies to cope with higher or lower than normal incubation temperatures. Overall, Madkour et al. (2021) claimed that thermal acclimation at the postnatal stage or throughout the embryonic stages has been considered as a novel promising strategy to mitigate the detrimental effects of heat stress in poultry. These authors suggested that, for large-scale application, this strategy needs further investigation to determine the suitable temperature and poultry age.

There is a loss in egg weight during incubation due to the evaporation of water (Rahn et al., 1977). This is crucial to make available ample air needed for the lung ventilation of the embryos sequel to internal pipping and ultimately hatching (Ar and Rahn, 1980). The best hatchability has been attained with 12–14% water loss at embryonic day 18 (Ar and Rahn, 1980). The relative humidity in the incubator can be manipulated to control the water evaporation of the eggs during incubation (Buhr, 1995). Lower or higher relative humidity could have variable effects on hatching and post-hatch performances.

Van der Pol et al. (2013), investigated the effect of low or high relative humidity and showed showed that incubating eggs at a higher or lower relative humidity negatively influenced hatching performances. They found that at the same incubation temperature (37.8°C), the reduction of relative humidity increased egg weight loss at E18 and reduced hatchability by increasing late embryo mortality. But it did not affect the chick’s weight, quality and hatching time. They found that incubating eggs at a low RH compared with a high RH and maintaining the EST at 37.8°C decreased the hatch of fertile eggs. Bruzual et al. (2000) reported that relative humidity of less than 63% during incubation decreased chick weight. Reducing relative humidity during incubation had little effect on post-hatch performance according to Van der Pol et al. (2013). El-Hanoun et al. (2012), investigating the effect of incubation humidity and flock age on hatchability traits and post-hatch growth in Pekin ducks found that the optimal relative humidity depends on breeders’ flock age.

It is well known that a temperature of 37.5–37.8°C and relative humidity of 55–60% are optimal environmental conditions for efficient embryo development, and the best hatching and post-hatch performances. Any change of one of these two factors without changing the second accordingly could have variable effects on incubation results. (Boleli and Queiroz, 2012) found an interaction of these two incubation parameters on hatchability. They also found a moderate negative correlation between hatchability and temperature (r = −0.41), hatchability and egg weight loss (r = −0.31) and a positive correlation between hatchability and relative humidity (r = 0.47). The effect of the interaction of temperature and relative humidity was also significant on incubation duration and chicks’ body weight.

Egg turning involves four major factors that can be taken into account; the position of eggs, angle of turning, frequency (times/day) and stage of incubation in whom this turning occurs. These different factors of turning diversely influence the physiology of the embryo, incubation parameters and post-hatch performances.

The lack of turning during incubation has been reviewed (Lundy, 1969; Baggott et al., 2002). A complete absence of turning during the first but not the second week of chicken eggs incubation leads to an increase in mal-positioned embryos and mortality (Elibol and Brake, 2004). The results of New (1957) showed that days 3 to days 7 of incubation were critical and failure to turn eggs during this critical period leads to a decrease in hatchability and rates of embryonic growth (Deeming, 1989). Moreover, the lack of turning of chicken eggs between days 12 and days 19 of incubation (last stages of embryonic development) leads to less embryonic growth as a result of impaired O₂ consumption through the chorioallantoic gas exchanger (Pearson et al., 1996). Tona et al. (2003a) noted that turning of eggs until 12, 15, and 18 d of incubation did not affect the levels of plasma corticosterone in the developing embryo or newly hatched chicks. The author inferred that corticosterone might not be involved in the mechanism by which turning affect hatching and production parameters. However, they observed that turning beyond 15 days increased pCO₂ in air cells (hypercapnia) and plasma levels of T₃ and T₄ at the internal pipping. In contrast to Tona et al. (2003b) who showed a correlation between this increase in metabolism, hatching time and hatchability, these authors observed that these parameters remain similar between eggs turned until 12 days and 18 days, suggesting the involvement of other intrinsic factors.

Egg turning at 90° and 45° on either side of the vertical using as a standard practice in the industry was linked to considerable research during the 1930s–1950s (Baggott et al., 2002). Lesser turning angles increase mal-positioned and embryo mortality in domestic fowl (Funk and Forward, 1953; Elibol and Brake, 2006; Cutchin et al., 2009). Schalkwyk et al. (2000) noticed that hatchability amounted to 1·83% for an increase of 1° (R ² = 0·96) when ostrich eggs were rotated hourly through angles ranging from 60° to 90°. In a recent study, Guo et al. (2021) recorded a shortened incubation duration and improvement in hatchability and goslings’ quality when eggs were turned at a wider angle (60° compared with 50°). They also observed an increase in late embryos and goslings’ weight correlate with a significant upregulation of genes in the somatotropic axis (GHRH, GH, and IGF-1 mRNA expression) and muscular development (pax7, MyoD, MYF5, and MRF4 mRNA expression). The authors concluded that wider angle turning made full use of the albumen content in goose eggs and recommend adjusting the angle of turning to the ratio of albumen in eggs according to avian species as previously stated by other researchers (Baggott et al., 2002; Elibol and Brake, 2006; Deeming, 2009).

Static incubation impairs the expansion of the area vasculosa during the critical period of sub-embryonic fluid production in the domestic fowl (Baggott et al., 2002). This absence of egg turning delayed the formation of extra-embryonic fluids and reduced rates of embryonic growth later in embryonic development (Deeming, 1989). Turning once an hour is commonly used in the industry concerning fowl eggs. The increase of this frequency up to 96 times daily does not significantly improve incubation results (Freeman and Vince, 1974). By contrast, lower frequencies decrease these results. Indeed, Oliveira et al. (2020) showed a decrease in hatchability due to a gradual increase percentage of early and late mortality with less turning frequency (Table 2). Elsewhere, a study conducted by Elibol and Brake (2006) showed that increasing turning frequency is a good way to reduce mal-positioned embryos associated with less turning angle during incubation. These data suggest possible interactions between the different factors of turning that are not well explored.

Moraes et al. (2018) incubated Japanese quail eggs in different positions (vertical position with the small end up, vertical position with the small end down, horizontal position) without turning and found that hatchability is best when eggs were set with the small end down although the outcome was similar to those set horizontally (65.3 ± 6.4% vs. 59.3 ± 9.2). In a 2 × 2 factorial design trial, Schalkwyk et al. (2000) recorded an increase in hatchability when ostrich eggs were set horizontally for 2 weeks and vertically for the remainder of the incubation period compared to those set vertically for the entire incubation period irrespective of angles of rotation (60° or 90°). Nevertheless, the results of these trials encourage the vertical setting despite having no obvious advantage over the eggs set horizontally then vertically with the appropriate angle of rotation. This is consistent with practices in the industry supported by the work of Funk and Forward (1960) who recorded better hatchability when fowl eggs are set vertically with their air sac up. This position prevents hatching failure due to mal-positioning or pipped eggshells on the narrow end.

Studies on the effect of these different factors on egg turning on post-hatch growth are not well documented. However, many hypotheses can be made based on the results of turning on incubation parameters. Then, Takeshita and McDaniel (1982) observed that although chicks from eggs in horizontal or vertical with small end up required less time to exhibit initial pips, they required longer to emerge from the shell than those in vertical with the small end down. Usually, the spread of hatching can affect the time of first feeding. Shortness of hatching windows when eggs were set with the small end down would lead to improved growth performances through early access to feed, which is crucial for post-hatch performances (Gaglo-Disse et al., 2010; Wang et al., 2014). In addition to short hatching windows, Guo et al. (2021) recorded an increase of goslings of high quality with a proper turning angle. According to Tona et al. (2003a), day-old chick quality and relative growth up to 7 days as well as slaughter performance are positively correlated (Figure 4). The authors concluded that the quality of chicks was better with an adequate turning angle (45°) because these chicks were able to use more nutrients to produce body mass tissue. An adequate turning angle could improve the feed efficiency of birds during the growth period.

Chicken eggs contain a small amount of carbohydrate (less than 1%) that supplies glucose, the most important source of energy needed for embryo growth (Starck and Rickelefs, 1998). This amount of carbohydrates initially available in the egg could not cover all the needs of the embryo until hatch. Therefore, glucose and glycogen are preferentially utilized as energy sources over lipids and protein because the limited oxygen available is mainly generated via gluconeogenesis and glycogenesis (Pearce, 1971). These carbohydrates were important for the final stage of embryonic development especially for pipping and chick emergence from the shell (Christensen et al., 1993; Moran, 2007).

Glucose inoculated in albumen at days 7 during organogenesis failed to improve hatchability while chicks’ weight was significantly improved at hatch (Salmanzadeh et al., 2012). In contrast, in ovo injection of carbohydrates (glucose, maltose) during the late stage of incubation (at days 17.5 or days 18) did not affect hatchability or newly chick weight (Ipek et al., 2004; Dos Santos et al., 2010, Eslami et al., 2014). However, Zhai et al. (2011) recorded a decrease in hatchability with glucose, fructose, sucrose or maltose in ovo injected at d 18.5 of incubation although body weight or body weight relative to set egg weights were significantly increased. A combination of carbohydrates (maltose, sucrose and dextrin) inoculated in ovo on day 19 also increase the bodyweight of newly hatched chicks (Tako et al., 2004). Thus, in ovo injection of carbohydrates at the appropriate time during incubation seem to be a good way to improve embryo growth and then chick weight at hatch. In addition, an insufficient amount of glycogen in late-term embryo forces the embryo to mobilize more muscle protein for gluconeogenesis until replenishing of glycogen reserves with the access of newly hatched chicks to feed (Vieira and Moran, 1999a,b; Moran, 2007). Uni et al. (2005) showed that injection of a solution containing β-hydroxy-β-methylbutyrate (a leucine metabolite) into the amniotic fluid of broiler embryos on day 17.5 spare the use of pectoral muscle leads to an increased body-weight at hatch. The in ovo inoculation of amino acids mixture seems to be also a good way to improve significantly chick weight at hatch if the amino acids used were identical to the amino acids pattern of egg protein (Al-Murrani, 1982). In chicken embryos, days 19 of embryonic development is an important point when the risk of lipid peroxidation is very high because tissues are characterized by comparatively high levels of polyunsaturated fatty acids. But at this time, the natural antioxidants level is not sufficient for innate protection. This risk is more when internal piping occurs with increasing oxygen availability as pulmonary respiration begins. Thus, low antioxidant status increases the embryo’s susceptibility to lipid peroxidation. However, in ovo injection of vitamins (A, B1, B2, B6, and C or E) at days 14 of incubation failed to improve hatchability and chicks’ weight at hatch (Nowaczewski et al., 2012; Goel et al., 2013). In contrast, extract from plants like Moringa oleifera, Nigella sativa, etc. are rich in carotenoids, an antioxidant naturally present in eggs, increase significantly hatchability (N’nanle et al., 2017; Oke et al., 2021). The advantage of some plant extract use is to concentrate different nutrients like vitamins and trace minerals (selenium, copper, zin, and iron) that act as co-factor of many enzymes involves in hatching success (Malheiros et al., 2012).

Administration of exogenous nutrients and other agents in ovo can advance the development of the embryo and post-hatch growth (Uni and Ferket, 2003). Some nutrients in ovo injected lead to an increase of body weights at hatch (Al-Murrani, 1982; Tako et al., 2004; Uni et al., 2005; Zhai et al., 2011; N’nanle et al., 2017). Like chick quality, hatching weight is a major predictor of marketing weight in chickens. Thus, advantages observed at hatch in the in ovo feeding treatment were maintained during rearing (Al-Murrani, 1982; Uni et al., 2005). This gain could be attributed to changes that occur earlier in the gut. For example, Smirnov et al. (2004) and Tako et al. (2004) showed an increased surface area, length and width of villus at hatch and 3 days after with carbohydrates inoculated at days 17.5 in chicken eggs. In the same way, manna oligosaccharides injected in ovo at days 17 resulted in the newly hatched chick with more mature enterocytes in the small intestine that can enhance digestive capacity and epithelial barrier (Cheled-Shoval et al., 2011).

Although chicks’ weight at hatch was unaffected, Joshua et al. (2016) demonstrated that in ovo inoculation of nano form of selenium (0.075 or 0.15 µg/egg) and zinc (40 µg/egg) significantly improve weight gain, body weight at market age and feed efficiency (only with nano form of Zn). This could be explained by the involvement of these minerals in immunity (McKenzie et al., 1998; Cardoso et al., 2007). Immunity was also improved by in ovo inoculation of probiotics and synbiotics which led to better post-hatch resistance against pathogens (De Oliveira et al., 2014; Sławinska et al., 2014) while prebiotics have been reported to increase the number of beneficial bacteria and promote their early colonization in the intestine of neonatal chicks (Tako et al., 2014).