Control measurements of cyclic motility in normoxia were made on a total of 83 chick embryos at five incubation time points: D10 (n = 18), D12 (n = 19), D14 (n = 18), D15 (n = 20), and D16 (n = 8). The control data were first examined for age-related motility changes, the control data having been obtained from the experiments with long embryo exposure to normoxia, and from the control values in normoxia that were obtained from the experiments in which embryos were exposed to hypoxia. The chick embryos reached stage 36 on D10 and stage 42 on D16. The mean embryo weight increased from 2.31 ± 0.15 g on D10 to 15.1 ± 1.26 g on D16 (Fig. 2A).

In control (normoxia), the embryonic motility displayed а cyclic character and each cycle consisted of an APh which was separated by an IPh (Fig. 2B). Long recordings (120 min) of embryonic motility in normoxia showed that the values of the average duration of APh and IPh at the beginning of exposure did not differ from their values at the end of exposure at all ages studied except Day 14 when the average duration of APh increased over the course of the recording (Fig. 2B).

The average APh duration significantly increased from D10 (30.5 ± 0.8 s; n = 18) to D12 (46.9 ± 1.2 s; n = 19) (P < 0.001) and remained constant between D12 to 15. During the next day, the APh duration significantly decreased (32.2 ± 5.4 s (n = 8), P = 0.008). The IPh duration significantly decreased in the period from D10 (42.8 ± 1.3 s; n = 18) to D12 (26.1 ± 1.0 s; n = 19) (P < 0.001), remained unchanged from D12 to D14 (n = 18; P = 0.15), then decreased until D15 (20.3 ± 1.1 s; n = 20) (P = 0.006), and remained constant until D16 (Fig. 2A). The mean durations of APh and IPh on D10 and D14 were comparable with our earlier results for these embryonic ages [17Nechaeva M, Vladimirova I, Alexeeva T. Effect of acute hypoxia on the motor activity and heart rate of the 10-and 14-day chick embryo. Open Ornithol J 2010; 3: 127-33.
[http://dx.doi.org/10.2174/1874453201003010127] ]. The average duration of the motility cycle that consisted of the activity phase duration plus the duration of the subsequent inactivity phase remained unchanged from D10 to D15, and then was decreasing until D16 (Fig. 2A).

Changes in the embryo weight during development shown in Fig. (2A) agree with the results of many previous studies, which confirm that the embryos grew at normal rates.

Our study of the changes in cyclic motility during the D10-16 period of incubation showed that the APh lengthened and the IPh shortened in the period from D10 to 12; then, neither APh nor IPh durations changed between D12 and 14; from D14 to D15, IPh duration decreased and APh duration remained unchanged (Fig. 2A). At the same time, the average duration of the activity - inactivity cycle which is defined as the sum of the durations of two consecutive phases (APh and IPh), remained constant from D10 to D15 (Fig. 2A). On D16, the average cycle duration was substantially decreased because the APh duration was considerably shorter than on D15 (P = 0.008), and the IPh duration remained unchanged. We studied the hypoxic effect on cyclic motility until D15 inclusive. Then the motility character was beginning to gradually change mainly due to the appearance of repetitive limb movements that have been described by Bradley et al. [23Bradley NS, Ryu YU, Lin J. Fast locomotor burst generation in late stage embryonic motility. J Neurophysiol 2008; 99(4): 1733-42.
[http://dx.doi.org/10.1152/jn.01393.2007] [PMID: 18272869] ], and the cyclic character of the motility became less pronounced. Other authors have also reported an increased variation of the parameters of cyclic motility (APh and IPh) with embryonic age [16Bradley NS. Age-related changes and condition-dependent modifications in distribution of limb movements during embryonic motility. J Neurophysiol 2001; 86(4): 1511-22.
[PMID: 11600617] , 24Ganley KJ, Bradley NS. Transformations in embryonic motility in chick: E9 to E18. Soc Neurosci Abstr 1999; 25: 2177., 25Rose D, Ganley K, Bradley N. Transformations in embryonic motility in chick: E9 to E15. Soc Neurosci Abstr 1998; 24: 1153.]. This prevents the use of these parameters as quantitative estimates of the motility after D15, especially in studying the effects of exogenous factors.

Age-related changes in the parameters of cyclic motility during the period studied (Fig. 2A) generally agree with the results of earlier studies [7Hanson MG, Landmesser LT. Normal patterns of spontaneous activity are required for correct motor axon guidance and the expression of specific guidance molecules. Neuron 2004; 43(5): 687-701.
[http://dx.doi.org/10.1016/j.neuron.2004.08.018] [PMID: 15339650] , 16Bradley NS. Age-related changes and condition-dependent modifications in distribution of limb movements during embryonic motility. J Neurophysiol 2001; 86(4): 1511-22.
[PMID: 11600617] , 17Nechaeva M, Vladimirova I, Alexeeva T. Effect of acute hypoxia on the motor activity and heart rate of the 10-and 14-day chick embryo. Open Ornithol J 2010; 3: 127-33.
[http://dx.doi.org/10.2174/1874453201003010127] , 26Wu KC, Streicher J, Lee ML, Hall BK, Müller GB. Role of motility in embryonic development I: Embryo movements and amnion contractions in the chick and the influence of illumination. J Exp Zool 2001; 291(2): 186-94.
[http://dx.doi.org/10.1002/jez.1068] [PMID: 11479917] ], where these parameters were also used for qualitative and quantitative estimation of the motility. However, note that Bradley [16Bradley NS. Age-related changes and condition-dependent modifications in distribution of limb movements during embryonic motility. J Neurophysiol 2001; 86(4): 1511-22.
[PMID: 11600617] ] and Wu et al. [26Wu KC, Streicher J, Lee ML, Hall BK, Müller GB. Role of motility in embryonic development I: Embryo movements and amnion contractions in the chick and the influence of illumination. J Exp Zool 2001; 291(2): 186-94.
[http://dx.doi.org/10.1002/jez.1068] [PMID: 11479917] ] found that the APh and IPh durations were close to each other on D10.

Our results have demonstrated a distinct dependence of the cyclic motility response to 20-min hypoxia during the D10-15 period of incubation on the embryonic age, the dependence being observed at both degrees of hypoxia studied (10 or 5% O₂) (Figs. 3 and 5). In all cases, the cyclic motility was decreased compared to normoxia (control). The changes in the average durations of APh and IPh during hypoxic exposure reflected the strength of the inhibitory effect on cyclic motility but did not allow detailed analysis of the events within hypoxic exposure. Therefore, it was necessary to trace the time course of these parameters. Stepwise measurements of the APh and IPh at 5-min intervals during hypoxic exposure and the subsequent recovery in air have revealed the details of cyclic motility changes during the hypoxia (Fig. 4). We have found that the time course of the cyclic motility response to hypoxia depends not only on the embryonic age (D10-15), but also the degree (10 or 5% O₂) and duration (20 or 40 min) of hypoxia.

The most important result of our research was the discovery of a new phenomenon, namely, the ability of a chick embryo to maintain motility in hypoxia, not only on D14 as we first discovered [17Nechaeva M, Vladimirova I, Alexeeva T. Effect of acute hypoxia on the motor activity and heart rate of the 10-and 14-day chick embryo. Open Ornithol J 2010; 3: 127-33.
[http://dx.doi.org/10.2174/1874453201003010127] ], but also at other developmental stages. In addition we succeeded in quantitatively measuring motility during hypoxia, studying its change with growth, and analyzing its dependence on the hypoxia level. The capacity of the embryo to maintain the cyclic motility under acute hypoxia was manifested, first, in the experiments under prolonged hypoxia, that is the experiments where the hypoxia duration increased twofold; and, second, the experiments in which the time course of the cyclic motility response to hypoxia was measured at 5-min intervals.

Thus, it was found that when the exposure to hypoxia was prolonged from 20 min to 40 min, on D12, the inhibitory effect on cyclic motility decreased in 10% hypoxia (average IPh decreased) and increased in 5% hypoxia (average IPh was increased and APh decreased) as compared to what was observed during the first 20 min of exposure (Figs. 3 and 5). At an older embryonic age (D15), the capacity of the embryo to maintain the cyclic motility under acute hypoxia was expressed to a larger extent and the inhibitory effect observed during the prolonged hypoxia (40 min) was weaker than that observed after the first 20 min at both levels of hypoxia, the weakening being more pronounced in 10% hypoxia (Fig. 5). While the mechanisms that maintain and even enhance embryonic motility are unknown, they are already effective on D12 in 10% hypoxia and on D15 at both levels of hypoxia studied.

The analysis of embryonic cyclic motility (duration of APh and IPh) during hypoxia also showed the embryo’s capacity to maintain the cyclic motility under 20 min 10% hypoxia. Embryonic movements first stop completely (thus IPh continues without any APh duration) and then begin to recover before the end of hypoxic exposure (APh and IPh occur alternately). We first observed this characteristic biphasic motility response to hypoxia (10 min 10% O₂) on D14 [17Nechaeva M, Vladimirova I, Alexeeva T. Effect of acute hypoxia on the motor activity and heart rate of the 10-and 14-day chick embryo. Open Ornithol J 2010; 3: 127-33.
[http://dx.doi.org/10.2174/1874453201003010127] ]; however, the duration of the exposure was insufficient for embryonic motility to be completely restored under hypoxia. The present study has shown that prolongation of the hypoxic exposure to 20 min at the same embryonic age (D14) leads to a complete restoration of cyclic motility during hypoxia (Fig. 4E).

A question arises as to the mechanisms underlying the recovery or maintenance of motility under hypoxia and the causes of its changes with embryonic age. Two main factors may determine it at each embryonic age: first, the degree of morphological and functional development of the embryo's motor system and the systems involved in oxygen supply to embryonic tissues; second, the maturation level of physiological regulatory mechanisms.

The involvement of these factors and their correlation can be traced in the incubation process. On D10, the biphasic motility response to 10% O₂ was not pronounced and the hypoxic effect on cyclic motility developed gradually (Fig. 4A). We assume that this response was slow because the system of oxygen supply to the embryo was not yet developed enough. This resulted from a small gas-exchange area on the egg surface on D10, when the chorioallantoic membrane (CAM) had not covered the entire egg [27Romanoff AL. The Avian Embryo: Structural and Functional Development. New York: Macmillan 1960.], as well as low blood flow rate [28van Golde J, Mulder T, v Straaten H, Blanco CE. The chorioallantoic artery blood flow of the chick embryo from stage 34 to 43. Pediatr Res 1996; 40(6): 867-71.
[http://dx.doi.org/10.1203/00006450-199612000-00016] [PMID: 8947964] ] and blood oxygen transport capacity [29Tazawa H, Andrewartha SJ, Burggren WW. Development of hematological respiratory variables in late chicken embryos: the relative importance of incubation time and embryo mass. Comp Biochem Physiol A Mol Integr Physiol 2011; 159(3): 225-33.
[http://dx.doi.org/10.1016/j.cbpa.2011.02.024] [PMID: 21377532] ] at this embryonic age. The considerable inertia of the hypoxic response on D10 was also expressed in the slow restoration of embryonic motility after the hypoxic exposure: the IPh duration continued increasing for 5 min after the hypoxic gas mixture had been replaced with air (Fig. 4A).

At later stages (on D12 and D14), hypoxia (10% O₂) caused the distinct biphasic response of cyclic motility. This was more pronounced on D12 than on D14, while the initial temporary cessation of embryonic motility under hypoxia was longer on D12 than on D14 (Fig. 4C, E). The initial arrest of embryonic motility under hypoxia was apparently caused by a rapid decrease in O₂ supply to the spinal neurons responsible for the rhythmic embryonic motility. This resulted from (i) an increase of gas exchange efficiency due to the growth of the CAM that had surrounded the entire egg contents by D11-12 [30Freeman BM, Vince MA. Development of the avian embryo: a behavioral and physiological study. London: Chapman and Hall 1974.
[http://dx.doi.org/10.1007/978-94-009-5710-7] , 31Gabrielli MG, Accili D. The chick chorioallantoic membrane: a model of molecular, structural, and functional adaptation to trans epithelial ion transport and barrier function during embryonic development. J Biomed Biotechnol 2010; 2010: 940741.] and development of the chorioallantoic vascular network; (ii) considerable acceleration of blood circulation [28van Golde J, Mulder T, v Straaten H, Blanco CE. The chorioallantoic artery blood flow of the chick embryo from stage 34 to 43. Pediatr Res 1996; 40(6): 867-71.
[http://dx.doi.org/10.1203/00006450-199612000-00016] [PMID: 8947964] ]; and (iii) an increase in the blood oxygen transport capacity [29Tazawa H, Andrewartha SJ, Burggren WW. Development of hematological respiratory variables in late chicken embryos: the relative importance of incubation time and embryo mass. Comp Biochem Physiol A Mol Integr Physiol 2011; 159(3): 225-33.
[http://dx.doi.org/10.1016/j.cbpa.2011.02.024] [PMID: 21377532] ]. The recovery of cyclic motility to the control level by the 20th minute of hypoxia may be accounted for by activation of regulatory mechanisms that were already sufficiently mature on D12-14. These may be, e.g., heart rate restoration against the background of hypoxia [17Nechaeva M, Vladimirova I, Alexeeva T. Effect of acute hypoxia on the motor activity and heart rate of the 10-and 14-day chick embryo. Open Ornithol J 2010; 3: 127-33.
[http://dx.doi.org/10.2174/1874453201003010127] ], changes in hematological respiratory variables [32Andrewartha SJ, Tazawa H, Burggren WW. Acute regulation of hematocrit and acid-base balance in chicken embryos in response to severe intrinsic hypercapnic hypoxia. Respir Physiol Neurobiol 2014; 195: 1-10.
[http://dx.doi.org/10.1016/j.resp.2014.01.019] [PMID: 24509299] , 33Tazawa H, Andrewartha SJ, Burggren WW. Acute regulation of hematocrit and blood acid-base balance during severe hypoxic challenges in late chicken embryos (Gallus gallus). Respir Physiol Neurobiol 2012; 184(1): 86-96.
[http://dx.doi.org/10.1016/j.resp.2012.08.002] [PMID: 22902513] ], and redistribution of blood flow towards the most important organs (including brain) in response to hypoxia, which is known to become more pronounced with embryonic age [34Mulder AL, van Golde JC, Prinzen FW, Blanco CE. Cardiac output distribution in response to hypoxia in the chick embryo in the second half of the incubation time. J Physiol 1998; 508(Pt 1): 281-7.
[http://dx.doi.org/10.1111/j.1469-7793.1998.281br.x] [PMID: 9490852] ]. These regulatory mechanisms may contribute to the restoration of motility under hypoxia. The mechanisms are more developed on D14 than on D12; therefore, the restoration of embryonic motility begins earlier, and the initial cessation of motility under hypoxia on D14 is shorter than on D12. If the hypoxia on D12 was prolonged from 20 to 40 min, the restored motility decreased again during the second half of the exposure (Fig. 4C). Apparently, the maturation level of the regulatory mechanisms underlying this recovery was not sufficient for maintaining the motility for a long time.

As development continued (D15), however, there was no distinct cessation of embryo movements at the beginning of hypoxic exposure, and the motility decreased due to a considerable prolongation of IPh and shortening of APh, which continued to occur alternately (Fig. 4G). It may be assumed that the regulatory mechanisms by this age were already mature enough to maintain the cyclic motility under hypoxia; hence, they were triggered more quickly and cyclic motility did not cease at the beginning of hypoxia. On the other hand, the APh and IPh were restored to the pre-hypoxic values only by the 35th minute of hypoxia, in contrast to D12 and D14, when the motility rate reached the control level by the 20th minute of hypoxia. Apparently, some additional factors hindered the rapid restoration of motility. For example, it was previously shown that the structure of the APh changes with age and it is determined by the activity not only of spinal neurons, but also the brain, muscles, and proprioceptors [10Hamburger V, Balaban M, Oppenheim R, Wenger E. Periodic motility of normal and spinal chick embryos between 8 and 17 days of incubation. J Exp Zool 1965; 159(1): 1-13.
[http://dx.doi.org/10.1002/jez.1401590102] [PMID: 5215365] , 16Bradley NS. Age-related changes and condition-dependent modifications in distribution of limb movements during embryonic motility. J Neurophysiol 2001; 86(4): 1511-22.
[PMID: 11600617] ]. The considerably greater decrease in the APh duration during hypoxia on D15 compared to earlier ages was probably accounted for by switching off of these additional components, which might have been restored under hypoxia more slowly than the activity of spinal motor networks.

The results of experiments with 5% hypoxia also confirm a high maturation level of the regulatory mechanisms on D15. In contrast to earlier embryonic ages, when such hypoxia rapidly led to an almost complete immobility of the embryo, the same exposure on D15 did not cause cessation of embryonic motility; moreover, there was a tendency towards motility restoration during the exposure, although its rate did not reach the control level.

The time period we indentified for the gradual development of the embryonic capacity to maintain motility under acute hypoxia is similar to the time course of development of some other embryonic capacities, including embryonic resilience to chronic hypoxia [35Dzialowski EM, von Plettenberg D, Elmonoufy NA, Burggren WW. Chronic hypoxia alters the physiological and morphological trajectories of developing chicken embryos. Comp Biochem Physiol A Mol Integr Physiol 2002; 131(4): 713-24.
[http://dx.doi.org/10.1016/S1095-6433(02)00009-0] [PMID: 11897182] ], and the period for changing the damaging hypoxia influence on the chick embryo brain [36Rodricks CL, Gibbs ME, Castillo-Melendez M, Miller SL. The effect of hypoxia on the functional and structural development of the chick brain. Int J Dev Neurosci 2010; 28(4): 343-50.
[http://dx.doi.org/10.1016/j.ijdevneu.2010.02.004] [PMID: 20171268] ]. Our results supplement the available data on the existence of the so called window in development when chick embryos are capable of compensating for the hypoxia effect [35Dzialowski EM, von Plettenberg D, Elmonoufy NA, Burggren WW. Chronic hypoxia alters the physiological and morphological trajectories of developing chicken embryos. Comp Biochem Physiol A Mol Integr Physiol 2002; 131(4): 713-24.
[http://dx.doi.org/10.1016/S1095-6433(02)00009-0] [PMID: 11897182] ]. Additionally, data concerning the development of the stress response during this same period of chick embryogenesis [37Epple A, Gower B, Ten Busch M, Gill T, Milakofsky L, Piechotta R, et al. Stress responses in avian embryos. Am Zool 1997; 37: 536-45.
[http://dx.doi.org/10.1093/icb/37.6.536] -39Tona K, Onagbesan O, Bruggeman V, Mertens K, Decuypere E. Effects of turning duration during incubation on embryo growth, utilization of albumen, and stress regulation. Poult Sci 2005; 84(2): 315-20.
[http://dx.doi.org/10.1093/ps/84.2.315] [PMID: 15742969] ], suggests that the gradual development of the embryo’s capacity to maintain motility under acute hypoxia parallels the development of the stress response. Consequently, the physiological compensatory response to acute hypoxia could develop during the period studied.

In summary, the results of the study indicate that the response of cyclic motility to acute hypoxia varies during D10-15 of incubation. The age-related changes in the responses to moderate (10%) and severe (5%) hypoxia differ from each other. In 10% hypoxia, the strongest inhibitory effect on the cyclic motility was observed on D15; in 5% hypoxia, the strongest effect was on D12, with severe hypoxia inhibiting cyclic motility more than moderate hypoxia at all embryonic ages except D15 when the effect of both levels of hypoxia was about the same (Figs. 3 and 5). We believe that the cyclic motility response to acute hypoxia at different embryonic ages is mainly determined by the strength of the inhibitory influence of hypoxia, on the one hand, and, by the capacity of the embryo to maintain cyclic motility upon a decrease in the О₂ concentration, on the other. The interaction between these two factors determines the degree of reduction of embryonic motility during hypoxia relative to the normal level characteristic of the same embryonic age. It is still unclear what regulatory mechanisms are involved in the hypoxic response. However, analysis of the changes in the parameters of cyclic motility under hypoxia at different developmental ages revealed that the embryo became capable of maintaining motility under hypoxia during D10-15. This capacity was increased with embryonic age, which we relate to the maturation of the regulatory mechanisms. For example, on D12, cyclic motility was partly restored during 10% hypoxia, but the regulatory mechanisms were not mature enough to maintain the motility under more severe hypoxia (5% О₂), where the motility stopped completely. By D15, the maturation of the regulatory capacity reached the level at which embryonic motility was maintained under both 10% and 5% hypoxia. Further studies are necessary in order to better understand the role of the regulatory mechanisms involved in the cyclic motor response to hypoxia at different stages of incubation.