The Difference of Metabolic Rate of Avian Eggs During Incubation

Chelsea Roche and Frank Leon Department of Biological Sciences

Saddleback College Mission Viejo, CA 92692

Metabolic rate is an important measurement of all of the body’s functions. It can be measured by oxygen consumption or by the production of carbon dioxide. Both methods of measurement encompass body functions such as gas exchange, organ function and the overall health of an individual. Metabolic rate increases as the gestational period goes on, and is also dependent on the size of the organism. In this experiment, the metabolic rate of avian eggs was measured in relation to the point in the gestational period. Measurements were taken at the beginning, middle -1 -1 and end of gestation and calculated in terms of mL CO2*g *min . The original experiment tested the metabolic rates of chicken, ducks and quail eggs against each other with relation to weight of the egg, but investigators encountered an experiment-altering problem in which the majority of the eggs were not fertilized, as they were supposed to have been. Investigators adapted and used the five quail eggs that were fertilized and developing to measure the metabolic rate in beginning, middle and end of the gestational periods. The average carbon dioxide production was calculated for comparison purposes. An ANOVA test was run, with a p-value of 0.046 followed by a Post-Hoc Bonferroni Comparison and it was found that there was a statistical difference between the middle and late gestational periods (t=2.807). The statistical difference was present because an unexplained event occurred where all of the fetuses died in-utero. There was no statistical difference in beginning to middle of the gestational periods (1.690), or beginning to end of gestation (t=1.117). Introduction

The process in which a bird broods its eggs is vital to the success of the population. The term brooding is defined as an inherent behavioral characteristic present in the class Aves to sit on a clutch of eggs to incubate them (Ekarius, 2007). The body heat from the presence of the parent provides a constant temperature that is necessary to promote embryonic growth and development. The heat present from incubation provides the energy needed to drive molecular interactions and enzymatic activities. Oxygen drives phosphorylation through a pressure gradient created by the embryo’s oxygen consumption. Carbon dioxide and water vapor diffuse in the opposite direction creating a metabolic process in which the embryo will grow from. Towards the end of incubation, breathing like movements begin and the beak pierces the air cell to fully develop the lungs (Mortola, 2009), marking the end of the dependence of the embryo on the egg for metabolic processes during development. The size of the egg and the incubation time are directly related. There is no significant direct relationship, however, to the size of the bird and the size of the egg. Smaller species lay relatively larger eggs in comparison of body size and egg size. The varying incubation times are significant in determining the maturity of the bird at hatching time. In altricial species, the hatchlings are completely dependent on parental care while precocial species that are born after longer incubation periods are born with their eyes open and are more developed than the former (Hoyt, 1980). All eggs used in this experiment are precocial species. Metabolism in developing embryos has been measured in the form of heat and oxygen consumption. The metabolic rate increased during development and plateaued just prior to hatching. Metabolic rate is proportional to egg mass and inversely proportional to incubation time (Karlsson, 2007). The incubation time for all three species varies. The common chicken’s incubation time is 21 days, the duck’s incubation time is 26-28 days and the quail’s incubation time is the shortest at 16-17 days. Investigators hypothesized that the duck will have the highest metabolic rate, and that metabolic rate will be at it’s highest just before pipping. This hypothesis is supported by numerous studies done on the metabolic rate of eggs based on size and the different points in the gestational period.

Materials and Methods Investigators obtained 12 fertilized duck eggs, 12 fertilized quail eggs and 12 fertilized chicken eggs from Wagon Train Tack and feed in Orange, California. An incubator and a turner were also rented to insure a good hatching. Water was placed in the incubator to keep humidity at 65%, the amount needed for all three species. The incubator was turned on and allowed to reach 37C. All eggs were placed in the turner, which would rotate the eggs every hour like they would be in the wild. This insures that the eggs do not get stuck to the shell, and are allowed to grow in equal proportions all around their bodies. The eggs were left for a period of a week in which temperature and humidity were checked every day. After a week, the first measurements were taken for all three species and divided thereafter by the time left in the gestational period and measurements were taken accordingly. The mass of each egg was taken and recorded before each measurement. Measurements were taken using a Pasco GLX probe, which measured the amount of carbon dioxide produced. Eggs were set in a sealed container, which was then placed in an incubator set at 37C. Carbon dioxide production was measured for ten minutes and graphed on the Pasco GLX. The data was then transferred to a laptop and graphed on excel to measure the slope and R2 value. The data was divided by 1000 to get the carbon dioxide in mL, divided again by the volume of the container used to capture data, and then divided yet again by the mass of each egg.

Results After encountering difficulties with fertilization of 31 of the 36 eggs, only five quail eggs were used to gather data, as they were the only eggs that underwent some sort of development. An ANOVA test was run to compare the averages of CO2 produced by the five eggs at three different gestational periods, beginning, middle and end with a p-value of 0.046 (Figure 1). According to the Post-hoc Bonferroni comparison, there were statistical differences between the middle and end of the gestational period (t=2.807), but not between the beginning and end (t=1.117), or the beginning and middle (t=1.690). The average of the beginning of all five eggs was 0.256 mL*g-1*min-1. The average of the middle gestational period was 0.274 mL*g-1*min-1 and the average of the late gestational period was 0.119 mL*g-1*min-1.

Figure 1: The average of the production of CO2 by the five quail eggs at beginning, middle and end of gestation.

Discussion The results indicated that there was a difference in metabolic rate in the early stages of avian development. The current experiment supported previous work (Mortola, 2009) that the metabolic rate of the avian egg size increased during the early and middle stages of the gestational period. An increase in carbon dioxide production indicated an increase in metabolism, likely due to the development of organs. Later in the gestational period, the production of carbon dioxide decreased, signifying a decrease in metabolic rate for all five quails. Investigators suspect that development was halted for an unexplained reason. All research and knowledge of metabolism of Avian eggs supports the theory that metabolism should be at it’s highest just before pipping, but this was not the case, indicating that something was wrong. After the hatch date had been passed by a week for the quails, investigators broke open portions of the eggs to find that 7 of the 12 quail eggs were not fertilized, and 5 had died unexpectedly during development. This prompted an investigation to the rest of the eggs in which the candle method was used, where a hole was cut in a dark piece of construction paper and a light was shone through it to see where the rest of the eggs were in their developmental stages. Investigators were shocked to find that 31 of the 36 eggs were unfertilized and not developing, rendering the original experiment impossible. Investigators adapted to the situation and changed the experiment to use the data that they had collected for the five quails during varying gestational periods. Instead of measuring avian egg size versus metabolism as the original experiment outlined, investigators measured the carbon dioxide production versus the period of incubation. It was found that carbon dioxide did increase, as it should, from the beginning to the middle, but at the end of gestation which was the only statistically significant comparison, according to the Bonferroni comparison, carbon dioxide production decreased significantly. Acknowledgements

Investigators would like to thank Steve Teh, for his knowledge and expertise. They would also like to thank their classmates for the moral support they provided for the duration of this experiment. Literature Cited

Birchard, G.F and Deeming, D.C. (2009). Avian eggshell thickness: scaling and maximum body mass in birds. Journal of Zoology 279: 95-101.

Ekarius, Carol (2007). Storey's Illustrated Guide to Poultry Breeds. 210 MAS MoCA Way, North Adams MA 01247.

Hoyt, Donald and Rahn, Hermann (1980). Respiration of Avian Embryos. Respiration Physiology 39: 255-264

Karlsson, Ola and Lilja, Clas (2007). Eggshell structure, mode of development and growth rate in birds. Zoology 111: 494-502.

Mortola, Jacob P. (2009). Gas Exchange in avian embryos and hatchlings. Comp. Biochem. Physiol. Part A 153: 359-377.