Coturnix japonica (Japanese quail) were used as a model organism to study the effects of maternal stress on prenatal development. Two quail lines were obtained that had been selectively bred for high or low corticosterone levels. Reciprocal crosses were made between the lines. 48 eggs total were incubated and embryos were measured from 7-16 days of development. Results showed that the high corticosterone line (HH) and low-high cross (LH) had similar growth rates, as did the low line (LL) and high-low cross (HL), indicating maternal corticosterone exposure influenced development more than direct genetics. Analysis of bone growth supported this, with femur and humerus growth consistent

1. References
Discussion
Glucocorticoids became a major focus during the 1970s and 1980s. Early
experiments showed how male rat offspring, from restraint stressed mothers,
showed masculinized and feminized sexual behavior. Subsequent studies
supported the hypothesis that high glucocorticoid levels, during the prenatal
stages of development, caused a shift in the timing of the testosterone peak.
This led to a desynchronization in the maturation patterns of testosterone
secretion (Ward and Wisz 1980). More specifically, stress and corticosterone
levels were found to have a direct relationship (Dahlöf and Hard 1978). These
early studies began to show the importance of corticosterone on prenatal
development.
Prenatal bone growth has been found to have an inverse relationship with
overall postnatal growth (Clum et al 1995). Several studies have used bone
measurement to generate models of growth in organisms. Femoral and
humeral lengths, specifically, are preferred measurements to indicate linear
embryonic growth. This technique can be applied to models of growth
focusing on factors such as stress.
Coturnix japonica has been a model organism in several fields, including
developmental biology and behavioral science. As with other model
organisms, several different genetic lines have been developed which express
certain genotypes, including factors such as plasma corticosterone levels. The
plasma corticosterone-controlled lines were developed by stressing the hens
before laying, as described by Hayward et. al. 2005. Stress is induced by
handling the hen, removing food and water for a short period of time, and/or
altering the normal social hierarchy for a short period of time. The stressed
hens have been shown to have higher plasma corticosterone levels, which
transfers to its eggs as result of its fat solubility. Stressed hens were used to
produce a high stress line by selection for those animals having the highest
plasma corticosterone level, HS, while those with the lowest corticosterone
response were used to produce a low stress line, LS (Hayward et al. 2005).
After 10 generations of selection, the lines exhibited high or low plasma
corticosterone without the application of the stressor. We hypothesized that
maternal stress will greatly influence the rate which quail embryos develop.
This effect should carry through generations, as well. We crossed the HS and
LS lines of quail to produce HH, HL, LH and LL lines. Hayward et al. noted
that levels of corticosterone to the egg in the egg yolk, are controlled
maternally. By comparing the reciprocal crosses LH and HL),can differentiate
between the maternal transfer of corticosterone to the egg (causing similar
developmental patterns in the HH and LH line) from either direct or sex-
linked (i.e., paternal) genetic effects.
Eggs of the species C. japonica were obtained from Dr. Nick B.
Anthony of the Department of Poultry Science, University of Arkansas.
The eggs were of two different strains, marked, Low Stress(LS), and High
Stress(HS). Reciprocal crosses were also made between the two strains
with the following combinations: HL, and LH. Each cross was done in the
order male, female. About 48 eggs were set in total in the Leahy
Manufacturing Co. incubator at 45 % humidity. The embryos were
incubated until they were ready to be dissected at certain ages. From the
point of being set until dissection, the eggs were rotated 180°, using the
genotype demarcation on the shell as a gauge. At ages 7-16 days-old, the
embryos were dissected and pictures of the embryos were taken by a Canon
camera. The pictures were then uploaded on to a computer, and measured
using the software imageJ. The embryos’ total size, humerus, femur, and
beak were measured. The measurements were then graphed and analyzed
using JMP software.
Methods and Materials
Coturnix japonica, the Japanese quail, has been a model organism in several fields,
including developmental biology and behavioral science. During the past few years we
have used quail as a model organism while investigating the effect of stress on prenatal
development. Two lines of quail were obtained from Dr. N. Anthony (University of
Arkansas) that had been divergently selected for high and low levels of plasma
corticosterone. It was observed that the high strain (HS) eggs hatched earlier and
developed at a faster rate, whereas the low strain (LS) hatched later and developed at a
slower rate. In this study we made reciprocal crosses between the two strains, in order
to differentiate between direct genetic and maternal effects (in this case, corticosterone
exposure) on embryonic development. We hypothesized that the maternal stress would
greatly influence the rate at which quail embryos develop. Forty-eight eggs in total were
incubated at 77% humidity and the developing embryos were removed between 7-16
days after incubation during their 18-day development period. The total length of the
embryo along with the femur, beak, and humerus lengths were measured at each age
using ImageJ software. The high (HH) strain had a similar growth rate to the low-high
(LH) cross, with logarithmic curves of log(y)=2.19±0.14x and log(y)=2.01±0.15x
respectively. Further, the low (LL) strain was observed to have a similar growth rate to
the high-low strain, with logarithmic curves of log(y)=2.55±0.11x and
log(y)=2.43±0.11x, respectively. In these results it was observed that rate of
development corresponded with the maternal strain in both crosses. Also, further
analysis suggested that the percentage of growth of the humerus and the femur
appeared to be consistent with the maternal lines. Both the HH strain and LH strain
were similar, and the LL strain and HL strain were found similar as well. The data
collected from this study supports that the embryo’s development is largely influenced
by the maternal effect and is not a genetic one.
Abstract Alex Torres, Rishi Patel, Melissa Cornelius
Faculty: Guy Barbato
Genetic analysis of prenatal skeletal development
of Japanese quail divergently selected for stress
responsiveness
Results
Figure 1
The graph shows the logarithmic growth curve differences in HH, LH, HL,
and LL based on their overall embryo length. Equations are shown on the
graph (A-D).
Table 1.
Contains the intercept and slope of the four lines. Intercept is initial length of
embryo, and slope is the growth rate of each line.
Table 2.
Contains the genetic effect that was calculated for evidence of maternal effect.
The pure lines were subtracted to get the line effect and the reciprocal crosses
were subtracted to get the reciprocal effect. Testing for heterosis was
calculated by subtracting the sum of the reciprocals from the sum of the pure
lines
Figure 2
The growth of the femur, humerus, and beak as the embryo developed from
ages 7-16 of the four different lines (A-C)
The results showed when looking at the total embryo length by age, the HH line
and LH line were seen to have relatively close initial lengths and growth rates. The
initial lengths of the HH line and LH line were 2.19mm±0.33 and 2.01mm±0.32. The
growth rates for the HH line and LH line were 0.14mm/day±0.03 and
0.15mm/day±0.03; both these lines show to have the highest growth rates. The LL and
HL lines also were seen to show relatively similar developmental patterns. The initial
lengths of the LL line and HL lines were 2.55mm±0.16 and 2.43mm±0.34; both these
lines show to have the highest initial lengths. The fact that the maternal strain in the
reciprocal crosses have similar initial lengths and growth rates as the pure lines show
the first piece of evidence that they are influenced by the maternal effect.
Further evidence, was seen when linear contrasts were performed in an attempt to
look at the genetic comparison. The line effect between the pure lines gave the
differences of -0.36mm and 0.03mm/day, the line effect was subtracted from high to
low. These results support our prior studies that showed that the low strains had higher
initial lengths since it was negative, and that the high stains were observed to have
faster growth rates, since the number was seen to be positive. The reciprocal effect
between the two reciprocal lines gave differences of -0.42mm and 0.04mm/day, the
reciprocal effect was subtracted from high to low based on the maternal strain. The
differences from the reciprocal effect were shown to go in the same direction as the
line effect; this shows the maternal effect in the reciprocal crosses.
Another calculation was done to test for heterosis which resulted to be 0.3mm for
the initial length and 0.01mm/day for the growth rate, since the values did not come
out negative, heterosis was not seen to have occurred. Heterosis was determined by
subtracting the sum of the reciprocal crosses from the sum of the pure lines, since both
sums contained the same amount of each strain, hypothetically they should have been
equal to zero. The growth rate’s value was relatively close to zero, the initial length
however had a much higher positive value. We are not certain why such a large value
was observed, however it may have been due to inconsistencies in measuring the
lengths of the embryos. In addition, when the graphs were created, the initial lengths
observed did not account for the first days of cellular development, the earliest
embryo measured was 7 days old.
According to a study done by Sissons HA and Hadfield GJ 1955 with rabbits on
cortisone it was observed that the longitudinal bones inability to grow was because of
the cessation of proliferating cells. This study supports why the HH and LH start off
initially smaller, and then have a growth rate greater than the LH and LL in our study.
The four lines were all observed to show a similar growth pattern when analyzing the
skeletal development. The growth of the beak was seen to do the majority of its growth
before the embryos were measured (before the age of 7 days). As a result, the growth
percentage based compared to the total body length was seen to decrease for all four
lines as the embryo aged. On the other hand, in all four lines, the femur and humerus
were observed to have relatively constant growth, meaning that they accounted for the
total growth measured in the embryos.
Figure 1: Total length of each embryo between the ages of 8 days to 16 days
old for the four lines (A-D), measured with ImageJ, and created with JMP.
Table 2: Comparison between crosses for maternal effect
quantitatively through genetic effect
Figure 2: Percentage of growth relative to total growth for the femur (A),
humerus (B), and beak (C) for the four lines of quail
Dahlöf ,L.G., Hård E, Larsson K. 1978. Sexual differentiation of offspring of mothers treated with cortisone during pregnancy. Physiol Behav
21(4):673-4.
Hayward LS, Satterlee DG, Wingfield JC. 2005. Japanese quail selected for high plasma corticosterone response deposit high levels of
corticosterone in their eggs. Physiol Biochem Zool 78(6):1026-31.
McEwen BS and Wingfield JC. 2003. The concept of allostasis in biology and biomedicine. Horm Behav 43(1):2-15.
Romero LM and Butler LK. 2007. Endocrinology of stress. Int. J. Comp. Psychol. 20: 89-95.
Saino N, Romano M, Ferrari RP, Martinelli R, Moller AP. 2005. Stressed mothers lay eggs with high corticosterone levels which produce low-
quality offspring. Journal of Experimental Zoology, Part A: Ecological Genetics and Physiology 303A(11):998-1006.
Sissons HA and Hadfield GJ. 1955. The influence of cortisone on the structure and growth of bone. J Anat 89(1):69-78.
Ward IL and Weisz J. 1980. Maternal stress alters plasma testosterone in fetal males. Science (New York, N.Y.) 207(4428):328-9.
Linear Contrasts Difference in
Intercept (mm)
Difference in
Slope
(mm/days)
HH-LL -0.36 0.03
LH-HL -0.42 0.04
(HH+LL)-(LH-
HL)
0.3 0.01
Crosses Intercept (mm)
+/- SD
Slope
(mm/day) +/-
SD
HH 2.19±0.33 0.14±0.03
HL 2.43±0.34 0.11±0.03
LH 2.01±0.32 0.15±0.03
LL 2.55±0.16 0.11±0.1
Table 1: The embryo’s initial size and
growth rate for the four lines.
Introduction