Aerobic scope in chicken embryos

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Abstract

We investigated the aerobic scope of chicken embryos, that is, the margin of increase of oxygen consumption (V̇O2) above its normal value. V̇O2 was measured by an open-flow methodology at embryonic ages E3, E7, E11, E15, E19 and at E20 at the internal (IP) and external pipping (EP) phases, at the normal incubation temperature (Ta = 38 °C), in hypothermia (Ta = 30 °C) and in hyperthermia (Ta = 41 and 44 °C). In the cold, Q10 averaged ~ 2 at all ages, except in IP and EP when lower values (~ 1.5) indicated some degree of thermogenesis. In hyperthermia (38–44 °C) Q10 was between 1 and 1.4. Hyperthermia had no significant effects on V̇O2 whether the results combined all ages or considered individual age groups, except in IP (in which V̇O2 increased 8% with 44 °C) and EP embryos (+ 13%). After opening the air cell, which exposed the embryo to a higher O2 pressure, hyperthermic V̇O2 was significantly higher than in normothermia in E19 (+ 13%), IP (+ 22%) and EP embryos (+ 22%). We conclude that in chicken embryos throughout most of incubation neither heat nor oxygen availability limits the normal (normoxic-normothermic) values of V̇O2. Only close to hatching O2-diffusion represents a limiting factor to the embryo's V̇O2. Hence, embryos differ from postnatal animals for a nearly absent aerobic scope, presumably because their major sources of energy expenditure (growth and tissue maintenance) are constantly maximized.

Introduction

In animals with no thermal control changes in ambient temperature (Ta) cause parallel changes in body temperature (Tb); these, then, are responsible for changes in oxygen consumption (V̇O2) according to the Q10 (Arrhenius) factor. The Q10 term is a quantitative approximation of the general concept that reactions rates depend on the absolute temperature; it expresses the change in velocity of metabolic processes for a 10 °C change in Ta, asQ10=M1/M210/T1T2where M1 and M2 are the speed of metabolic reactions at the corresponding temperatures T1 and T2. Around physiologically relevant temperatures, the rate of common chemical reactions increases by a factor of ~ 2 for every 10-degree Celsius change in temperature. At the organismal level, therefore, in absence of Tb-regulation a 10 °C change in Ta approximately doubles V̇O2; that is, metabolic Q10 = 2 (Bennett, 1984, Elias et al., 2014). Q10 values for V̇O2 < 2 indicate some effectiveness in the control of Tb. In the cold, the stability of Tb despite the drop of Ta implies the presence of effective thermogenesis, in which case, metabolic Q10 < 1. In the heat, the stability of Tb in the face of a rise in Ta (i.e., metabolic Q10 = 1) indicates an efficient control of heat loss.

Avian embryos have almost no thermogenic capacity, which, in fact, initiates only close to hatching. Indeed, measurements in various species including the chicken embryos have shown that both Tb and V̇O2 drop as Ta decreases, with Q10 ~ 2 (Romanoff, 1972, Nair et al., 1983, Williams and Ricklefs, 1984, Tazawa and Rahn, 1987, Tazawa et al., 1989a, Feast et al., 1998, Nichelmann et al., 1998, Whittow and Tazawa, 1991, Mortola and Labbè, 2005, Mortola, 2006).

By comparison to the response to cold, the embryo's metabolic response to heat has received less attention, although prenatal hyperthermia has biological interest and carries clinical implications. During pregnancy, fever or other conditions of a modest rise in Tb can have serious consequences on fetal development (Edwards, 1967, Smith et al., 1978, Edwards et al., 2003, Power and Blood, 2011). Mammalian experimental models to the study of prenatal hyperthermia have been used, but the uterine, maternal and placental responses to changes in Ta complicate the interpretation of the effects of hyperthermia on the fetus; furthermore, maternal stress during pregnancy by itself impacts on fetal development (Fernandez-Cano, 1958, Hensleigh and Johnson, 1971, Saetta et al., 1988, Tazumi et al., 2005). Avian embryos are free from some of the confounding factors typical of mammalian preparations; hence, they have been the experimental models for many studies, mostly concerned with small increments in incubation temperature on embryonic growth. The increase in Ta (by 1–2 °C) at various times in incubation have produced mixed results, with accelerations of embryonic growth (Hammond et al., 2007, Collins et al., 2013), decreases (Yalçin and Siegel, 2003, Yalçin et al., 2008, Piestun et al., 2009) or no significant changes (Iqbal et al., 1990, Moraes et al., 2004, Yahav et al., 2004, Yalçin et al., 2005, Ipek et al., 2014, Krischek et al., 2016). The scatter of results probably originated from the transversal protocols on separate groups of embryos, compounded by the large normal variability in embryo's development (Mortola and Al Awam, 2010).

Only a handful of studies have considered the effects of hyperthermia on embryo's V̇O2. In chicken embryos incubated at 39.5 °C for several days V̇O2 was slightly higher, similar or lower than in controls (37.8 °C) depending on the embryo's age (Piestun et al., 2009). Higher temperatures have not been tested, possibly because incompatible with survival when protracted for a long fraction of incubation (Romanoff, 1972). A brief (3 h) exposure to 39 °C of chicken and duck embryos in the second half of incubation raised V̇O2 by < 5% (Janke et al., 2002); higher temperatures were not tested. In another study, a progressive increase of egg temperature (5 °C in 2 h) in chicken embryos just before or during the internal pipping stage had, respectively, no effect or a 13% increase in V̇O2 (Bícego and Mortola, submitted for publication). Younger ages have never been tested. Hence, we do not have enough information on the embryonic Q10 during hyperthermia, and whether it varies throughout incubation.

Based on the information currently available, several possibilities can be anticipated. First, a rise in Ta may increase V̇O2 with a Q10 = 2. This result would imply that the amount of heat available to the normothermic embryo limits its V̇O2 (“heat-limitation”). A second possibility is that the rise in V̇O2 with Ta occurs only in the presence of additional oxygen. This possibility (“O2-limitation”) stems from previous observations in normothermic embryos close to hatching, in which exposure to hyperoxia increased V̇O2 (Høiby et al., 1983, Burton and Tullett, 1985, Tazawa et al., 1992a, Dzialowski et al., 2007). With respect to oxygenation in hyperthermia the only previous study was on turtle eggs; it showed no effect of hyperthermia on embryo's body mass or hatching success either in normoxia or hyperoxia (Liang et al., 2015). A third hypothetical result should contemplate the eventuality that V̇O2 remains unaltered after lifting both the O2- and the heat-limitation. Such absence of aerobic scope would imply that in normothermia the embryo's metabolic processes are working at maximal power with no room for further acceleration of the cellular biochemical functions (“power-limitation”).

The purpose of this study was to quantify the potential limitations imposed by the two essential substrates (heat and oxygen) on the aerobic scope of chicken embryos at various ages of incubation. To this end, first, we have measured the Q10 of the V̇O2 response to hypothermia; then, we have compared to it the data of acute hyperthermia before and after opening the eggshell region above the air cell, an intervention that lifts the barrier to O2 diffusion (Nakazawa and Tazawa, 1988). The results have revealed that of the three possibilities listed above the third one occurs most frequently throughout incubation; that is, the embryo's metabolic activity constantly operates close to its maximal value.

Section snippets

Methods

Freshly laid chicken (Gallus gallus domesticus, layer line) eggs were purchased from a local supplier. They were weighed individually and at midday (embryonic day 0, E0, out of 20.5 days of total incubation) were placed in a still air incubator (Hova-Bator model 1602, Savannah, GA, USA) set at 38 °C temperature, 60% relative humidity and automatic 90° egg rotation four times per day. Measurements were conducted at E3, E7, E11, E15, E19 and at E20 on embryos that had entered the IP (internal

V̇O2 in normothermia, before and after air cell opening

Embryonic V̇O2 increased as incubation progressed (Table 1); the values in normothermia were like those reported previously on chicken embryos for eggs of ~ 60 g of fresh weight (Mortola and Al Awam, 2010). The opening of the air cell produced no significant changes when embryos of all ages were combined (− 1% ± 4; N = 41) or when they were individually considered by age, except in the IP groups, when V̇O2 increased by 8% ± 4 (N = 6, P < 0.02; Table 1 and Fig. 1).

Q10 in hypothermia and in hyperthermia

The average hypothermic Q10 (38–30 °C range)

Discussion

The results indicated that the Q10 for V̇O2 in hypothermia was close to 2 at all embryonic ages except during hatching (IP and EP), when it was lower. In hyperthermia, Q10 was always less than in hypothermia and often close to 1; in fact, hyperthermia caused a significant rise in V̇O2 only in IP and EP. Finally, opening the air cell to increase the O2 diffusive conductance had insignificant effects other than in embryos close to end-incubation (E19, IP).

Conclusions

Hyperthermia and oxygenation have nearly no effects on the chicken embryo's V̇O2 for most of incubation, except toward end-incubation (E19, IP, EP); even at these late ages, the rise in V̇O2 is small by comparison to what expected for ectothermic animals. The low hyperthermic Q10 does not imply that thermoregulatory (heat dissipation) mechanisms are efficient. Rather, the most likely interpretation is that the embryos are operating at the highest V̇O2 compatible with cell survival, to maximize

Acknowledgements

Satoko Tomita Ide was a postdoctoral research fellow on leave from the Department of Dental Anesthesiology, Tokyo Dental College, Tokyo, Japan. Ryoji Ide was a postdoctoral research fellow on leave from the Department of Physiology, Nippon Dental University, School of Life Dentistry at Tokyo, Tokyo, Japan.

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