Unveiling the Consequences of Hypoxia and Thermal Stress in Avian Embryos: The Crucial Role of Pore Density

Abstract

The effects of anthropogenic global warming attributed to greenhouse gas emissions and urbanization are a significant concern for ecologists. Increasing ambient and surface temperatures have been shown to negatively impact the survival and physiology of various species, which can ultimately affect their population size or even lead to extinction over time. Many studies focus on understanding how cold-blooded (ectothermic) species such as fish, amphibians, and reptiles respond to increasing temperatures due to global warming, as they heavily rely on ambient temperatures to regulate their core body temperature. Avian species are unique in that even though they exhibit endothermic traits in adulthood, the avian embryos exhibit ectothermic phenotypes for most of their development. In addition to the ectothermic traits of avian embryos during development, their mode of respiration is also crucial for proper growth and survival. Unlike most mammals, avian embryos are separate from the maternal body and must rely on the microscopic pores that reside on their eggshell surface to respire essential gasses: oxygen (O2), carbon dioxide (CO2), and water vapor. Since these pores play a critical role in embryonic survival and development, several studies have explored whether the number of pores and the active pore area of the shell could be adjusted based on the environment avian mothers experienced to better prepare their embryos for the anticipated conditions. These studies have indicated that avian mothers can indeed adjust their eggs' active pore area, both decreasing and increasing it. However, it still needs to be well understood whether these changes positively affect embryonic survival and development. In Chapter 2, we conducted an experiment to investigate the role of avian eggshell pores in embryonic survival, water loss, and oxygen consumption rates. We covered 30% of zebra finch (Taeniopygia guttata castanotis) eggshells with all-natural beeswax to block the pores and then exposed them to either the control (37.4°C) or high (38.9°C) incubation temperatures. The lowest hatching success (or survival) rate was observed within the group that received both the wax and high incubation temperature treatments. In contrast, the control group that did not receive either of the treatments showed the highest hatching success rate. Regardless of the incubation temperature, the wax-treated groups showed reduced water loss and oxygen consumption rates compared to non-wax-treated groups. However, interestingly, the difference in oxygen consumption rates between the wax- and non-wax-treated groups became more apparent as development progressed. We also investigated the cause of embryonic mortality by opening the shell two days after the expected hatch day. Some unhatched individuals had a physical anomaly in the dorsal neck region, later identified as edema. In the subsequent chapters, I further investigate the cause of their mortality and the physical malformation. The main goal of Chapter 3 is to determine whether the aforementioned physical anomaly is a genuine malformation that occurred during incubation, as opposed to a postmortem occurrence due to a decaying body. To test this, we exposed zebra finch eggs to the same conditions used in Chapter 2 (wax treatment, control, and high incubation temperatures), but we did not allow the eggs to hatch naturally. Instead, we euthanized the embryos the day before they were expected to hatch to examine any physical anomalies. We found that both wax treatment and high incubation temperature led to edema formation in some zebra finch embryos, with the group that received both treatments showing the highest rate of edema formation. The edema was localized to the dorsal region of the embryo's neck, and we confirmed that it increased the head mass relative to the body by measuring the head-to-body ratio. Furthermore, we found that embryos with edema had a lower hue value in the color of their hearts, indicating a darker color. In Chapter 4, we focused on evaluating the body and organ masses of the hatchlings (from Chapter 2) and embryos (from Chapter 3) to determine if they are affected by elevated incubation temperature and/or the reduction in active pore area. We also examined any changes in embryonic body and organ masses and whether they are reflected in the hatchlings. Our findings showed that hatchlings in the high incubation temperature group had smaller body mass and higher yolk mass compared to those in the control incubation temperature group, but this trend was not observed in embryos. These differences in hatching and embryonic mass suggest that embryos with smaller body mass perform better under a high incubation temperature environment. Additionally, both hatchlings and embryos in the high incubation temperature group had smaller hearts. In Chapter 5, we histologically investigated the occurrence of edema in the dorsal region of the neck and various organs to determine if it is caused by failure or damage in specific organs. Our analysis revealed that individuals with edema had significantly lower glycogen levels in their liver. Furthermore, individuals with severe edema showed a higher incidence of hepatic necrosis and thinner ventricular walls in the heart than those without edema. The chapters of this dissertation emphasize the important role of microscopic eggshell pores in embryonic development and survival, as well as the exchange of essential gases through these pores. They also investigate potential physiological anomalies that may arise when the active pore area is reduced. By addressing the significance of eggshell pores, this research underscores the trade-offs and limitations in adaptive responses to increasing temperatures in non-true oviparous species and their eggs.