Hatching performance and chick weight may be influenced by the process of egg formation, which is affected by various factors such as egg weight (EW), egg quality, yolk nutrients, yolk antioxidant concentrations and variations in embryonic development (Surai et al., 2016). Eggs of different weights have different physical and chemical characteristics that affect embryo development depending on the concentrations of proteins, lipids, vitamins and antioxidants in the egg yolk (Burley and Vadehra, 1989).

Antioxidants are responsible for maintaining antioxidative protection in the tissues during embryonic development (Surai, 1999). Lipid-soluble antioxidants, such as vitamins A and E and carotenoids, are predominantly transferred to the developing embryonic tissues from the yolk sac during incubation (Surai et al., 1996; Surai, 1999). The yolk and the yolk sac membrane (YSM) contain high concentrations of vitamin A (Gaál et al., 1995), vitamin E, and carotenoids (Surai et al., 1996). The accumulation of vitamins A and E and carotenoids corresponds to extensive liver metabolism and decreases free radical formation (Surai et al., 1996). In the living organism, all antioxidants operate in cooperation with each other to form an integrated antioxidant system. The cooperative interaction of antioxidants in the embryonic cells is vital for the maximum protection of the embryo from the detrimental effects of free radicals (Surai, 2002). Oxidised vitamin E can be converted back into the active form by reacting with other antioxidants (Bohm et al., 1997). However, the effect, concentration and metabolism of these lipid-soluble antioxidants are dependent on dose, age (Khan et al., 2012), tissue and species (Surai, 2002; Karadas et al., 2011).

Vitamin E is the major player in antioxidant defence at the cellular and subcellular levels (Surai et al., 2016) and is known to be the most effective natural lipid-soluble and chain-breaking antioxidant. Structural analyses of vitamin E have revealed that there are four tocopherol molecules (α, β, γ and δ) and four tocotrienols (α, β, γ and δ) with antioxidant activity. Surai et al. (1996) reported that more than 80 % of vitamin E in embryonic tissues is represented by the most active form, α-tocopherol. Other isomers of vitamin E are found in the egg yolk and embryonic tissues, but at much lower concentrations. Furthermore, α-tocopherol exhibits its highest biological activity during the development of the embryo (Surai et al., 2016). Ionov et al. (1994) reported that the concentration of α-tocopherol in the egg yolk of quail was 252.4 µg g-1. The α-tocopherol level in the yolk sac increases by 3–5 times during the last week of incubation (Surai, 2002). Vitamin E is absorbed from the residual yolk sac (RYS) by the YSM and is then transferred to the liver during the prenatal development period of the avian embryo. The liver is the major site of vitamin E accumulation in the avian embryo. Thus, vitamin E accumulation in the liver during embryonic development is considered an adaptive mechanism in response to stress conditions during the hatching process (Surai, 2002). The chick embryonic brain is characterised by levels of vitamin E that are approximately 100-fold lower than those in the liver (Surai et al., 1996), and this apparent deficiency in the brain's antioxidant defence capacity is further compounded by the relatively low levels of vitamin A and carotenoids (Speake et al., 1999). In this study, tissues were selected according to their roles in lipid assimilation (liver), their polyunsaturated fatty acid (PUFA) content (brain) and their storage deposits (RYS) at hatching.

Vitamin E in the maternal diet can have profound effects on the health status, growth and development of the newly hatched chick. However, Hossain et al. (1998) observed that in ovo (IO) injection of vitamin E was more efficient in increasing the immune response of chicks than vitamin E supplementation to the maternal diet. Therefore, the aim of this study was to evaluate the effects of EW and IO α-tocopherol on eggshell temperature during incubation, hatching performance, chick development and lipid-soluble antioxidant concentrations in quail chick tissues at hatching.

This study was approved by the Van Yüzüncü Yil Üniversitesi Animal Use and Care Committee (permit number 2017-01).

In this experiment, yolk and wet eggshell weights and eggshell surface area values increased significantly with increasing EW (above 11.76 g). These results are consistent with results of Ashraf et al. (2016). Ashraf et al. (2016) also reported a lower yolk pH in lighter eggs than in heavier eggs, which is inconsistent with our results. The results of this study showed that the newly hatched weights of chicks from heavier eggs were significantly greater than those of chicks from lighter eggs every 4 h at the six measured time points. Egg weight increases in a linear manner with increasing breeding age as described by Yannakopoulos and Tserveni-Gousi (1987), and it has a direct influence on chick development. Due to increasing breeder quail age, significant variation in chick weight was observed from day 72 (EW between 8.21 and 11.75 g) to day 128 (EW between 11.77 and 14.46 g) of breeder quail age. As EW increases, heavier eggs begin to exhibit a higher eggshell surface area than lighter eggs, as reported by Shafey (2002). Lower amounts of egg water loss per square centimetre of surface area of eggshell were exhibited by heavier eggs compared with lighter eggs; however, this difference did not contribute to differences in embryonic mortality in the current study. The main reason for this is that average daily water loss did not differ among the groups because pore concentration, conductance and thickness of the eggshell remained similar among the EW groups. In the present study, the absolute and relative RYS weights were increased in heavier eggs, which is consistent with Peebles et al. (2000). In addition, the increased absolute liver weight and higher eggshell temperatures of the embryos from heavier eggs during late embryonic development might be related to differences in embryonic metabolism. This assumption is supported by O'dea et al. (2004), who found higher metabolic activity in heavier chicks; however, in our results, a lower RYS was observed in chicks from lighter eggs, reflecting the more rapid utilisation of nutrients. Moreover, in this study, eggshell temperatures were greater for the embryos of IO α-tocopherol-treated lighter eggs than those of similarly treated heavier eggs at days 9 and 11 of incubation. These results indicated that the IO injection of α-tocopherol into the yolk sac might have enhanced the metabolism of embryos from the light quail eggs by affecting the transfer of α-tocopherol from the yolk sac to the tissues before hatching. However, hatching time was prolonged in the α-tocopherol-treated chicks from the light quail eggs. This prolonged hatching time might have been due to high sensitivity of the embryos of lighter eggs to IO administration of α-tocopherol. This high sensitivity might be associated with the slightly higher plasma corticosterone concentration of chicks at hatch (Babacanoğlu and Yalçın, 2014).

The chick and middle toe lengths were the greatest for chicks that received IO α-tocopherol administration on day 5 of embryo development. These results indicate that IO α-tocopherol administration had positive effects on chick quality without affecting the developmental stability of the measured bilateral morphological traits. However, shank length was affected negatively by IO α-tocopherol administration, although its RA value was unaffected in the newly hatched quail chicks. This study is the first report on the effects of IO α-tocopherol injection on chick length and the RA values of the shank, middle toe and face lengths of quail at hatch. Middle toe length was decreased in the chicks from heavy eggs, whereas its RA was not affected by EW; however, the highest mean RA and the RA of shank length were observed in the chicks from light eggs in this study. The effects of EW on the RA of shank length and mean RA indicated that developmental instability of shank length can be promoted by a decreased egg weight in hatched quail chicks. Our results showed that there was no effect of IO α-tocopherol injection on hatchability. This result is consistent with Schaal (2008), who obtained a similar result with IO vitamin E injection. Our findings indicated that the total retinol concentrations of RYS and liver were higher in the heavier eggs, whereas the total carotenoid concentrations in all tissues and RYS were lower in the heavier eggs. These findings might be a result of vitamin A deficiency in the heavier eggs. Similarly, Hudon (1994) reported that carotenoids in heavier eggs could be converted to vitamin A. The low total carotenoid concentrations in the RYS and embryo tissues from heavier eggs might reflect the fact that total carotenoids can be efficiently converted to vitamin A (retinol), as reported by Burley and Vadehra (1989). Therefore, the newly hatched quail chick may convert appropriate carotenoids to vitamin A in the liver and RYS.

In this study, IO α-tocopherol injection into the yolk sac of the large egg increased α-tocopherol and vitamin E concentrations in brain tissues. The accumulation and concentration of lipid-soluble antioxidants in embryonic tissues may have adaptive significance in providing protection to PUFAs against peroxidation, particularly at hatching (Surai, 2002). It has been reported that large amounts of vitamin E accumulation may occur with a high PUFA in the lipid fraction of brain; therefore, lipids play an important role in vitamin E solubilisation and subsequent absorption (Surai, 2002; Surai et al., 2016). This observation was confirmed by Schaal (2008), who concluded that chicks injected with vitamin E had increased relative brain weight and brain total lipid level to protect lipid membranes from the harmful effects of radical oxygen species and allow for increased lipid utilisation for energy production.

The cooperative interaction between antioxidants in the cell is vital for maximum protection from the detrimental effects of free radicals (Surai, 2002). Oxidised vitamin E can be converted back into the active reduced form by reacting with other antioxidants, such as carotenoids (Palozza and Krinsky, 1992; Bohm et al., 1997). Increasing the dietary carotenoid concentration of the maternal diet was associated with increased vitamin E concentrations in the egg yolk and embryonic tissues and their increased resistance to oxidative stress (Surai and Speake, 1998; Surai et al., 2003, 2016). Therefore, we hypothesised that IO administration of α-tocopherol might increase the carotenoid concentration in embryonic tissues. However, IO α-tocopherol injection did not affect the total carotenoid concentration in the tissues of quail chicks in the present study. Our result is consistent with Frigg et al. (1992), who concluded that the carotenoid concentration of the yolk was unaffected by dietary α-tocopherol. Thus, neither dietary nor IO administration of α-tocopherol affected the carotenoid concentrations of chick tissues, with no effect of α-tocopherol on the carotenoid concentrations in the RYS in the present study. Similar results were reported by Possenti et al. (2017), who tested the effect of in ovo vitamin E on the distribution and use of other antioxidants and on oxidative status markers in the yolk and focal organs. They found that embryo growth was positively associated with antioxidant concentrations; however, an experimental increase in egg vitamin E level did not affect the relationships between other antioxidants or the markers of oxidative status and vitamin E concentration. In addition, in the present study, the highest vitamin E and α-tocopherol concentrations occurred in the order of RYS < liver < brain. Surai (2002) reported that the highest α-tocopherol concentration was found in the liver and that the lowest was found in the heart. This tissue specificity of α-tocopherol accumulation demonstrates that the location (yolk, albumen, amnion) and timing (embryo age) of α-tocopherol injection might affect reduced heart and liver weights following the injection of α-tocopherol on day 5 into the yolk sac in this study. Moreover, the reduced liver weight observed for the α-tocopherol-treated chicks at hatch was likely due to lower fat metabolism in these chicks relative to controls.

The results indicated that IO α-tocopherol injection might improve embryonic metabolism without affecting chick development in light quail eggs, whereas it might increase the concentrations of α-tocopherol and vitamin E in the brain tissues without affecting hatching performance in chicks from heavy quail eggs. In conclusion, IO α-tocopherol injection into the yolk sac and EW can affect the lipid-soluble antioxidant-specific and tissue-specific concentrations of lipid-soluble antioxidants in the tissues of newly hatched quail chicks.