Significant increase in liver and heart mass found in post hatching red junglefowls (Gallus gallus).

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Linköping University | Department of Physics, Chemistry and Biology Type of thesis, 16 hp | Educational Program: Physics, Chemistry and Biology Spring or Autumn term 2021 | LITH-IFM-G-EX—21/4032--SE

Significant increase in liver and

heart mass found in post hatching

red junglefowls (Gallus gallus).

Frederik Junge Pedersen

Examinator, Per Jensen Tutor, Jordi Altimiras

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1. Abstract

Significant heart and liver increase in hatching and neonates has been found evident in multiple different species such as the emu, Peking Duck, and pigs. The relative mechanism behind heart growth could be tied to closing of the ductus arteriosus, however there is still a debate whether it’s significant impact in avian species. Liver and heart mass were measured at four different transition stages before and after hatching on Red junglefowls (Gallus

gallus). Heart mass was found to vary between 83-170mg, with the lowest values most often

found in pre pipped chickens while higher values found more often in hatchlings. The relative heart mass was found to be significant (P<0,05) across all groups except between internally pipped and externally pipped chickens. Therefore, the results can conclude that an increase in relative heart mass was found to be directly tied to age. Liver mass was found to be

significant (P<0,05) but changes in mass was found to occur only between EP and hatchling stage. Further analysis on absolute heart and liver mass showed in both cases hatchlings having a higher mean mass compared to the other three stages. Both absolute liver and heart mass was found to be statistically significant (P<0,05) which indicates there are no major differences between analysis on relative or absolute mass. These findings suggest that red junglefowls follow the same growth pattern found in other precocial birds such as emu or Peking duck.

2. Introduction

The transition from egg or placenta to the outside is the final step in embryonic development. In the pre-neonatal stage, heat is supplied by the mother and oxygen diffuses through the placenta in mammals or the chorioallantoic membrance (CAM) in birds. Blood circulation in embryos bypasses the lungs through an embryonic vascular shunt which is known as the ductus arteriosus (DA) (Shell et al. 2016). The DA shunts blood away from the pulmonary arteries into the systemic arteries providing nutrients and oxygen to the systemic organs (Shell et al. 2016). In Red junglefowls (Gallus gallus), breathing starts at day 19-20 by internal pipping (IP) which then proceeds to external pipping (EP) just before hatching. This transition from oxygen diffusion via the CAM to convection of air in the lungs and alveolar diffusion causes the DA to close which results in an overload on the left ventricle (Beinlich et al. 1998).

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Evidence points to the fact that closing of DA stimulates a faster growth rate in the left

ventricle which results in larger heart mass in pigs and emus (Beinlich et al. 1998; Satoh et al. 2001; Shell et al. 2016).

What distinguishes birds from mammals is that the transition period from egg to breathing atmospheric air takes longer time. A study on heart mass in sheep show that there is an fast change in left ventricular mass once born (Snelling et al. 2019). However, a similar change in increased heart mass can be seen in emus, but the transition for breathing already starts in IP with the biggest change occurring during EP, leading up to hatching (Shell et al. 2016). The reason for the longer transition time is because birds start as ectothermic. They first become endothermic after hatching. This requires a functional cardiovascular system capable of pumping enough oxygenated blood to the tissues (Price & Dzialowski, 2017).

Birds also differ in maturity depending on species. They can be independent (precocial) or dependent on parental help (altricial) (Price & Dzialowski, 2017). Even though the timing of the onset of endothermy differs, they will still express similar functions related to

endothermic phenotypes (Price & Dzialowski, 2017). In the precocial Pekin duck, heart mass increases significantly during external pipping to post hatching (Sirsat et al. 2016). What exactly occurs heart growth can be seen in multiple different species, according to Shell (2016), the heart growth in sheep and emu occurred mainly from an increase in smooth muscle cell growth and a hypoxia induced response which promotes apoptosis. Whether hypertrophy or hypoplasia could be a contributor to increased heart mass, hypoplasia was found to be one of the leading causes of expansion of the heart in the Domestic White leghorns (Clark et al. 1989). In the Peking Duck, Sirsat (2016) showed that liver growth increased with age during pre-pipped to hatchling phase. However, the biggest increase was found to occur after hatching phase which Sirsat argued to be due to the liver having a role in increasing the basal or resting Vo2 of the hatchling (Sirsat et al. 2016).

These findings suggest that even though there is a difference in the transition phase between mammals and birds, drastic changes are still made to the development of the heart and

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internal organs before and after birth (Shell et al. 2016; Snelling et al. 2019). In this study, I will take a specific look at the Red junglefowls which is the ancestor to all domestic chickens and see if they follow similar growth patterns found in other species.

The goal of this study is to see if the heart, liver and brain increases in mass in relation to the time of hatching or birth as has been found in other species. I therefore hypothesize that we will see a similar organ development found in other precocial bird species and that the biggest change in mass occurs between EP and post hatching.

3. Materials and methods

3.2 Study subjects and classification into hatchling phases

The chickens were provided by my supervisor as surplus animals from previous studies. Animals were euthanized by injection with pentobarbital (100 mg/kg) after a minimum of 19d of incubation. In total there were 250 chicken embryos or hatchlings, 9 domestic White Leghorn and 241 Red Junglefowl. All eggs or hatchlings were weighed before sorting them into groups in relation to their hatching phase. They were categorized as pre pipped, internally pipped, externally pipped or hatchlings. To decide the hatching phase I tapped the blunt side of the egg and looked if the beak had pierced the CAM or not. If not, they were classified as not internally pipped (no IP). If they were internally pipped, they were classified as such (IP) unless the eggshell was pierced. In the latter case they were classified as externally pipped (EP). Animals were classified as hatchlings if they were euthanized after leaving the eggshell completely. In total, 210 Red Junglefowls were dissected. The distribution of our samples was found to be that No IP had 39, IP had 42, EP had 41 and Hatchlings had 84 samples in each group.

3.3 Test study

Nine White Leghorn hatchlings were used in a test study to train the dissection procedures. Based on the test study, the brain was excluded from future dissections because the brain tissue lost its consistency after thawing and it was not possible to retrieve all brain tissue consistently.

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4 3.4 Defrosting and handling of chickens

Samples were kept frozen at -20 °C. Samples were thawed based on a fixed time schedule. Hatchlings were placed in a refrigerator overnight (16 h) and kept at room temperature for 3 h. Chicken eggs were thawed in the refrigerator for 19 h and stored for 4 hours at room temperature during the day. In this way dissections could be performed when samples were still cold so the dissected organs could be frozen again for future studies.

Hatchlings were dissected and the liver, heart and yolk sac were removed. Each of the organs was weighed individually to the nearest one tenth of a gram (100mg). Chicken embryos were removed from the eggshell and weighed with their yolk sac still attached. The liver, heart and yolk sac were later dissected and weighed individually.

3.6 Statistics

Organ masses were calculated in relation to yolk-free embryonic mass, which was obtained by subtracting yolk mass from total embryonic mass.

A one-way ANOVA model was used to see if there was a difference between the ratio of heart mass and liver mass to total embryonic mass. A Tukey test was performed to see which means of groups in our ANOVA model was significant. A value of P<0,05 was used for all statistical analysis and regarded as a significant value. Only 206 samples were selected for statistical analysis and data from four samples were excluded as outliers. The reason for the deviating values could not be determined.

4. Results

4.1 Body Mass and Yolk Mass at different hatching phases

The rage of embryonic mass found in our data sample was between 13,3g to 28g. The distribution of my data samples was found to be hatchlings leaned more towards small egg size while No IP, IP and EP had a larger egg size. This can be seen by the distribution of embryonic mass found in figure 2. In figure 1 most of my hatchlings were found to be on the

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lower end of embryonic mass scale. Due to my samples being nonrandom but instead collected and provided by my supervisor, different analysis measurements were used to test both relative and effective mass on both liver and heart. Since my hatchlings were

overrepresented on mass intervals 13,3g – 18,00g, I decided to test relative heart mass which can be seen in figure 3 and 4.

Even though the distribution of small and large embryonic chickens varied, yolk sac mass still showed a clear difference between hatching phases as shown in figure 1. As expected,

hatchlings had the lowest amount of yolk mass independent of total embryonic mass. EP was found to have the second lowest followed by IP and No IP having the highest amount of yolk mass.

Figure 1 relationship between the amount of yolk sac mass compared to total embryonic mass found in four different age groups H (Hatchlings), EP (Externally pipped), IP (Internally pipped), No IP (Pre pipped)

4.2 Heart and liver mass at different hatching phases

In figure 2, the heart mass was found to be the highest across all samples of embryonic mass in hatchlings but had a lower mean heart mass compared to EP (Table 1). EP and IP chickens were found to have similar heart mass across all samples of body mass but differed in mean

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heart mass by 13mg. In No IP chickens, the heart mass was found to be the lowest with a mean heart mass of 98,46mg.

Table 1 showing mean value of HM (heart mass), LM (liver mass), HM:EM (relative heart mass), LM:EM (relative liver mass).. Each value is represented by the age of the chicken. HM:EM and LM:EM are both presented as a percentage.

Age Mean HM Mean LM Mean HM:EM Mean LM:EM

No IP 98,46 536,84 0,45518 2,4990

IP 113,119 573,35 0,50464 2,5714

EP 126,04 606,780 0,5215 2,5138

Hatchling 125,584 650,78 0,65731 3,3887

The liver mass showed a similar growth pattern found in figure 2. Hatchlings were found to have the largest liver across each mass. Highest liver mass in hatchlings were found to be 1200mg and the smallest was 530mg with a mean of 650mg. pre pipped chickens had the lowest liver mass across each embryonic mass with a mean of 536,84mg. EP and IP chickens showed no significant difference in liver mass, but the mean differed 23mg between them (Table 1). Both heart and liver mass showed similar growth patterns where hatchlings had the highest mean heart- and liver mass to embryonic mass ratio (table 1). The mass was found to be lower during earlier development stages.

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Figure 2 showing the relationship between heart- and liver mass compared to body mass excluding the yolk. The weights were measured in mg. Each categorical type describes at which stage the chicken belongs to. Type 0 = No IP (n=39). Type 1 = IP (n=42). Type 3 = EP (N=41). Type 4 = Hatchlings (n=84). Total chicken sample, N=206.

In figure 3, heart mass to embryonic mass ratio increased exponentially after each hatching phase. During No IP to EP, occurred an increase of 0,07% in heart mass compared to body mass ratio (Table 1). In EP compared to hatchlings, the heart to body mass ratio were found to drastically increase and differed 0,13% (Table 1). My ANOVA model showed P-value of 0,00 with a 95% significance.

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Figure 3 showing a one-way ANOVA test to measure the difference between each type of chicken. Y axis describes the heart mass to embryonic mass ratio. X-axis describes what type of chicken it is. Type 0 = No IP, N=39. Type 1 = IP, N=42. Type 3 = EP, N=41. Type 4 = Hatchlings, N=84. Total chicken sample, N=206. P<0,05. Each datapoint is the mean value of all the collected data samples.

In figure 4, the liver mass to embryonic mass ratios showed a significant increase from EP to Hatchlings and differed by 0,8%. Liver mass ratio showed no difference between No IP and EP chicken embryos. A drop in liver mass ratio was seen between IP and EP chickens but was deemed to be negligible. Our ANOVA model showed P-value of 0,00 with 95% significance.

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Figure 4 shows the ratio between liver mass and embryonic mass compared to each categorical type. Y-axis describes the liver mass to embryonic mass ratio. X-axis describes what type of chicken it is. Type 0 = No IP (n=39). Type 1 = IP (n=42). Type 3 = EP (n=41). Type 4 = Hatchlings (n=84). N=206. P<0,05. Each datapoint is the mean value of all the collected data samples.

4.3 Liver and heart mass on groups of embryonic mass

In the previous figures heart- and liver mass were presented as relative masses. In figure 5 and 6 I wanted to also look at the difference in absolute heart mass. For these purposes,

individuals were distributed to different bins depending on their embryonic mass as follows: Light group = interval 17,95 – 21,06. Middle group = interval 21,07 – 24,00. Heavy group = interval 24,00 – 27. Reasons why my intervals differed in mass size was because I wanted to get a better distribution from each transition stage. Mass intervals 13,3g – 17,95g was excluded due to insufficient sample size from each transition stage which can be seen in figure 2. The number of individuals in each weight group and transition phase is shown in Table 2.

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Table 2 Number of individuals in each weight group and phase for absolute heart mass

Phase Light group Middle group Heavy group

No IP 11 9 4

IP 10 16 7

EP 4 9 22

H 25 17 11

Looking at figure 5, group 1 showed a clear difference between each transition stage with No IP having the lowest mean absolute heart mass (88,5455g) and hatchlings having the largest (123,32g).

In middle group, there was still found to be a difference between No IP and hatchlings, however, IP had a larger mean (114,625g) compared to EP (113g).

In heavy group, the absolute heart mass was found to have the largest mean value in

hatchlings (151,182g). In this mass interval, EP now showed a larger mean compared to IP, however, No IP seemed to show a larger mean heart mass compared to IP.

For absolute heart mass, light and middle group showed a statistical significance (P<0,00) while group 3 showed a statistical significance (P=0,048) which can be seen in figure 8-10 (appendix). Between each transition stage only No IP and IP were statistically significant from hatchlings in light group (Figure 11 appendix). All transition phases were significant to hatchlings in middle group (figure 12 appendix), while no significant difference between groups were found in heavy group (figure 13 appendix).

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Figure 5 shows the absolute heart mass in three groups based on embryonic mass interval from figure 2. Each bar indicates the mean absolute heart mass from each transition stage.

Looking at figure 6, light group mean absolute liver mass was found to be highest in hatchlings (664g) while No IP had the lowest mean mass (490,83g). IP was found to have a higher mean mass (563,125g) compared to EP (528,25g). In table 3, the number of individuals in each group based on transition phase can be seen for absolute liver mass.

Table 3 Number of individuals in each weight group and phase for absolute liver mass

Phase Light group Middle group Heavy group

No IP 12 20 4

IP 8 25 7

EP 4 14 23

H 24 17 11

Middle group showed hatchlings having the highest mean absolute liver mass (685,118g) which is a slight increase compared to hatchlings in the light group. The mean between No IP, IP and EP showed close to no difference between them however, all transition stages did have a higher mean liver mass compared to the light group.

0 20 40 60 80 100 120 140 160 180 17.95g - 21.06g 21.07g - 24.00g 24.00g - 27g

Mean heart mass in different embryonic mass

intervals

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Heavy group had the highest mean absolute liver mass in hatchlings (887,91g). Each transition stage showed a continuous increase in absolute liver mass. Compared to the

previous groups, No IP had a lower mean liver mass (531,75g) compared to middle group, but still larger than the lighter group. IP and EP showed an increase in liver mass compared to both light- and middle group.

Figure 6 shows the absolute liver mass in three groups based on embryonic mass interval from figure 2. Each bar indicates the mean absolute liver mass from each transition stage.

Absolute liver mass was found to be statistically significant (P<0,05) across all three groups which can be seen in figure 14-16 (appendix). The difference between each group were found to be statistically significant only between hatchlings and the three transition stages. No significant difference was found between No IP, IP, and EP in all three weight groups (Figure 17-19 appendix).

5. Discussion

5.1 The heart

The results gathered in figure 2 indicates that there is a significant increase in mass of the heart from hatching stages. When chicken embryos transition from No IP to Hatchlings, we

0 200 400 600 800 1000 1200 17.93g - 21.06g 21.07g - 24.00g 24.00g - 27g

Mean liver mass in different embryonic mass

intervals

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can see in figure 2 that there is a distinction between each stage, showing that transitioning from diffusion to bulk flow induces hypertrophy on the heart. The biggest increase was found during EP to hatchling (figure 3) which confirms my hypothesis that there is a change in mass during embryonic development. The transition from No Ip to IP seen in figure 3 provides evidence for closing of the DA causing increased heart mass (Beinlich et al. 1998; Satoh et al. 2001; Shell et al. 2016). This could be tied to similar findings in emu that closure of the ductus arteriosus causes sudden increase in heart mass (Shell 2016). However, the closing of the DA occurs mainly between EP and hatchlings in emus, while in figure 3 indicates that the increase already occurs during IP. These findings are more like what has been found in other species of chickens (Rahn et al. 1985). Exactly why this is the case is something I can only speculate on, but since Red Junglefowls are the ancestors to all chickens, this is something I should have expected in my hypothesis. However, this still contributes to interesting findings in the evolutionary history on heart growth. Red Junglefowls being the ancestor to all

chickens provides phenotypic evidence to further strengthen the deviation from other avian species.

5.1.1 Absolute heart mass

In figure 7 (appendix), the difference between each group can be found to be statistically significant across all ages except between IP and EP (type 3 & 1). I expected there to be a higher difference between IP and EP because of previous findings by Rahn 1985, However my results only show minor increasing of heart mass in figure 3. In my previous figures, I have mainly looked at relative heart mass due to uneven distribution of large and small eggs. In figure 5 I managed to compare between hatching phases in absolute heart mass. In all three mass intervals, hatchlings were found have a larger mean absolute heart mass compared to the three previous transition phases. This is concurrent with evidence found on relative heart mass and was expected. However, there seems to be no general pattern in absolute heart mass between each mass intervals (figure 11-14 appendix). For instance, IP has a larger mean value (114,625g) compared to EP (113g) in middle group interval. This was not expected, but since we are looking at absolute heart mass, the difference in egg size could be determining factor. Either way, figure 7 (appendix) shows no difference between IP and EP in relative heart mass which should be similar when comparing absolute heart mass.

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My method analysis could be improved by having a more evenly distributed sample size from each transition stage. In figure 5 the distribution was very uneven which is one of the reasons I decided to look at relative heart mass in the first place. With more samples and more evenly distributed, I would have expected a clearer result similar to what have been found in figure 2, 3 and 4. However, both absolute and relative heart mass was found to be statistically

significant and therefore there is no major difference in analysis methods. 5.1.2 Thermoregulatory mechanisms and heart growth

Looking at figure 1, the yolk mass does not significantly decrease between IP and EP which could lead to a stalemate in growth during this transition. It could also be that the heart is increasing sufficiently enough to deal with the sudden change from diffusion to bulk flow. IP chickens are still inside the egg and are still ectotherms, so the heart could be increasing for the purpose of circulatory functions, and not thermoregulatory functions (Sirsat et al. 2016). It has been found that the heart pressure increases during later embryonic stages which could indicate the preparation in hatching chicks for the development of endothermic

thermoregulatory mechanisms (Girard, 1973; Sirsat et al. 2016). As we can see in figure 3, the relative heart mass increases the most from EP to hatchling transition stage. This is where the expected change in circulation is most prevalent since the atrial foramen and DA starts to close (Dzialowski et al., 2011). I mentioned previously that heart mass increase was found to be highest in the left ventricle in emus (Shell et al., 2016). This pressure change puts a lot of strain on the vessel walls. However, baroreflex functions have been shown to fully develop during late-stage embryonic development (day 18) to countermeasure the increasing pressure put on the heart during pre-pipping to internal pipping stage (Altimiras & Crossley, 2000). The maturation of the baroreflex continues to occur 3 days post hatching (Altimiras & Crossley, 2000), Girard (1973) showed that 3 hours post hatching the heart rate decreases significantly while mean pressure continues to increase. This could be a prevention for hypertension on the heart while still allowing the chick to develop endothermic mechanisms. 5.1.3 Closing of the ductus arteriosus and what we can do next.

Exactly how the increase in heart mass occurs in Red Junglefowls could tie into Sirsats (2016) discoveries where a significant increase in mass occurred on the left ventricle, however it is noted that there was an increase in right ventricle as well. When closing of the DA occurs in

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emus, Shell (2016) found that the lumen of the DA closes during IP. Due to a hypoxia induced response once the DA constricts, internal elastic lamina detaches from the

endothelium and a composition of smooth muscle cells starts to close the DA shut (Shell et al. 2016). What was found in neonatal pig heart is that closing of the DA causes an overload on the left ventricle wall to increase its wall thickness (Beinlich et al. 1998), but Sirsat (2016) argues that current evidence for avian species is unknown and begs the question whether her findings on increased ventricle mass occurs mostly in right, left or both sides at once.

However, these findings suggest that Red Junglefowls could see an increase in ventricle mass during late embryonic stage and that the left ventricle should follow similar patterns found in both pigs and emu. This is something that can be further studied on from my findings on increased heart mass.

5.2 The liver

Besides looking at the heart, I also wanted to see if other organs shared similar growth patterns. What figure 4 shows is that the increase in liver mass also occurs during EP to post hatchling stages. The biggest difference between the liver and heart is that the liver does not increase during the early stages of chicken development. There is no increase found in No IP, IP to EP stages which could indicate that the importance of the liver is not as high compared to the heart during early chicken embryo development. A livers functions is to process yolk, produce bile and excretion among other things and therefore the size of the liver could be sufficient to perform its intended function during early embryonic development. In figure 4, the difference is summarized by a one-way ANOVA test which gave us a p-value (P<0,05) and strengthens our assumptions that the difference during embryonic development is statistically significant. These findings share similarities with Sirsats (2016) on the Peking Duck where a significant increase in liver mass occurring during pre-hatching stages. However, liver growth occurred already in pre-pipped chicks while Red Junglefowls saw an increase during EP to hatchling. These findings still confirm my hypothesis but liver increase during EP could be related to yolk utilization and difference in allocation of resources

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16 5.2.1 Absolute liver mass

Looking at figure 6, the absolute liver mass was found to be highest in hatchlings across all group intervals. As expected, there was no significant difference between No IP, IP and EP across all three group intervals (figure 18-20 appendix). However, the ANOVA test still found that absolute liver mass does in fact show a significant difference across all means (Figure 15-17 appendix). These results show a similar response to relative liver mass found in figure 4 and there is no difference found between each analysis methods.

5.2.2 Externally pipped to hatchling.

The increase in EP to Hatchling could be tied to red junglefowls being a precocial species, however this is just a speculation and something I think would be interesting expanding further upon. The need to be independent requires body functions to be fully functional which could be why we see a drastic increase in liver mass. The blood in the right atrial of the heart could be the deciding factor in determining the increase in mass compared to the liver. To change from diffusion to bulk flow is a large change which requires significant structural changes to the embryonic heart. There is no known structural change to the liver during embryonic development like the shunt found in the heart, which could be the determining factor as to why the increase in mass occurs earlier in the heart compared to the liver.

Yolk utilization is also a factor that could be determining the significant increase occurring between EP and hatchlings. During its final moments between EP and hatching, the chick is unable to eat which requires nutrients from the yolk to be digested and consumed on a higher basis. According to Reidy (1998), poults in pre- and post-hatching chicks utilized all yolk contents which lead to a significant decrease in total yolk solid mass. Poult mass was also found to be the highest at the time of hatching, Reidy further suggested that substantial metabolized yolk content occurred three days before post hatching (Reidy et al. 1998). Future studies on yolk utilization may be deemed significant in how these organs increases in Red Junglefowls.

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17 5.3 The brain

My intentions for the study were also to look at the brain. What the test study showed was that the brain lost consistency after thawing. This made it hard to dissect it consistently and my supervisor and I decided to focus mainly on the liver and heart.

However, it can still be possible to speculate what the results could have been by comparing to mammals and other precocial bird species. There is evidence in multiple species indicating a sudden increase in brain growth during and after birth in mammals (Dobbing & Sands, 1979). This moment is defined as the brain growth spurt and similar growth pattern can be found in emus (Shell et al. 2016). During pre- to post-neonatal stages, the endothelial vascular shunt closes, which increases blood flow to the pulmonary arteries. It was because of this change that an increase in mass occurred to the heart (Shell et al. 2016). What we found in red junglefowls was that they shared similar heart growth patterns with other precocial birds and mammals (Shell et al. 2016). By looking at data found in figure 8, the amount of blood flow decreased to the brain and heart through the right-to-left shunt as the ratio of microspheres decreased (Shell et al. 2016). The brain follows a similar blood distribution pattern as the heart during neonatal stages. Since the brain is one of the major important organs, this could be a clear indication that the brain would follow a growth pattern like the heart found in figure 2. Since blood flow dictates nutrition and oxygen supply, the closing of the endothelial

vascular shunt provides an issue. Less blood is now going through the systemic arteries. Findings from Girard (1973), where heart rate was found to increase during closing of the DA could be a reason embryo solve this issue to meet the demand for brain growth after the transition from No IP to IP. I also mentioned yolk utilization found in poults saw a significant decrease in mass three days before hatching (Reidy et al. 1998). This could also contribute alongside increase heart rate a way for chicken embryos to increase brain mass. However, these are only speculations and there are probably a multitude of different factors contributing to increasing mass during pre- and post-hatching stages.

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Figure 8 Shows the change in blood flow during embryonic development in emus. Measurement on Chorioallantoic membrane (CAM), Heart and brain were conducted and deemed significant (P<0,05). A decrease in ratio is an increase in pulmonary flow through the pulmonary artery connected to the heart. E49: Embryo Day 49. IP: Internally pipped. EP: Externally pipped. Day 0: Hatchlings. Reused with permission from the publisher (Shell 2016)

5.5 Concluding remarks.

My aim with this study was to see if the heart and liver mass increased during pre- to post-neonatal stages in red junglefowls. Similar findings had been found and researched upon in other mammals and in precocial bird species such as Peking duck or emu. The purpose behind this study was to see if similar findings could be found in other species or if red junglefowls differed from precocial bird species. What I found was that age had a large effect on liver and heart mass. During transition from No IP to Hatchling, drastic changes occurred to heart mass. However, the liver only saw an increase in mass during EP to Hatchling. This could further be expanded upon that the closing of ductus arteriosus is the reason for the early increase in heart mass which was found to be similar in previously studied species. What I would like to have improved were to increase my sample size and distribute it more evenly when it comes to egg size and transition stage. Either way, my findings still conclude that heart and liver mass does increase during early embryonic development to hatchling. Going forward, studies on which chamber increases the most during each transition stage seems to be the next step in looking

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at growth rate in Red Junglefowls. Also, why the difference in heart mass in IP to EP were found to be the lowest increase between all transition stages could be an interesting note. I mentioned earlier that it could be due to circulatory functions and not thermoregulatory functions. However, the work I have presented have laid the groundwork for future studies related to heart and liver mass increase in red junglefowls.

6. Societal & ethical considerations

This study contributes to the agenda of the Linköping University for sustainable development. Knowledge of this study contributes to the scientific community and provides better education for all. It could provide significance in chicken embryo development and contribute to the pharmaceutical industry in the cardiovascular field of study.

Expanding further on the pharmaceutical industry, it is rather difficult and highly unethical to practice research on human embryos. From the top 22 research & development (R&D) countries, there is currently a practice of a 14-day limit on human embryos which provides limitations in R&D (Kristin & Moralí, 2020). This limitation prevents studies being made on for example heart and liver growth during fetal and neonatal stages. Therefore, model species are a good replacement which can be related to expanding human and veterinary medicine. Looking at my study on heart growth, the importance for further hemodynamic functions is of great interest for both the academic and pharmaceutical industries. It can be used to provide improved diagnostic tools for prevention of liver and heart diseases.

With improved diagnostic tools, intervention may be able to prevent earlier diseases and malfunctions by operating in utero. However, this leads to ethical dilemmas where effects on the mother may be detrimental to her health. It also poses the question whether preventions should occur in utero or after childbirth. The benefit of operating on fetal hearts is that they are easier to change compared to neonatal hearts.

The reason why chicken embryos function as a valid candidate for biomedical research is due to the chick being accessible and economic (Rashidi & Sottile, 2009). Larger animals are facing more ethical protests and their usage both technical and practical is limited (Rashidi &

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Sottile, 2009). The chicken on the other hand provides multiple benefits in both economical and practical usage and may function as a perfect candidate for in vivo studies (Rashidi & Sottile, 2009). However, usage of chickens may still lead to ethical dilemmas. For instance, multiple animal organizations want a permanent ban on animal usage in pharmaceutical industries which could spark a debate whether the importance of advancement in human medicine is more important than animals’ wellbeing. Either way, steps are taken to prevent harm and unnecessary stress put on testing animals which puts a more ethical method of dealing with model species.

With the use of animals in this study, careful procedures had been applied to minimize waste. My chicken embryos were euthanized by my supervisor under ethical conditions. All waste was stored in a container and put in the incinerator. The dissections were performed alone so that nobody could see in case of severe sensitivity.

7. Acknowledgements

I would like to thank my supervisor Jordi Altimiras for making this study possible and guiding me through my first big project. This was by far the highlight of all the things I have done, and I am very grateful for this opportunity.

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8. References

Altimiras, J., Crossley, A. D., 2000. Control of blood pressure mediated by baroreflex changes of heart rate in chicken embryo (Gallus gallus). American journal of physiology. 278(4), R980 – R986

Beinlich, C.J., Vitkauskas, K.J., Morgan, H.E., 1998. Characterization of ventricular myocytes from the newborn pig heart. J.Mol.Cell.Cardiol. 30, 1263-1274

Clark, E.B., Hu, N., Frommelt, P., Vandekieft, G. K., Dummett, J.L., Tomanek, R.J., 1989. American Journal of Physiology: Heart and circulatory physiology. 257(1), H55-H61. Dzialowski, E.A., Sirsat, T., Van der sterren, S., Villamor, E., 2011. Prenatal Cardiovascular Shunts in amniotic vertebrates. Respiratory physiology & Neurobiology. 178, 66-74

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9. Appendix

PRIMARY WORKSHEET

One-way ANOVA: HM:EM versus Type

Method

Null hypothesis All means are equal

Alternative hypothesis Not

all means are equal

Significance level α = 0,05

Equal variances were assumed for the analysis. Factor Information

Factor Levels Values

Type 4 0; 1; 3; 4

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Type 3 1,413 0,471048 81,15 0,000 Error 202 1,173 0,005805     Total 205 2,586       Model Summary S R-sq R-sq(adj) R-sq(pred) 0,0761889 54,65% 53,98% 53,04% Means

Type N Mean StDev 95% CI

0 39 0,45518 0,05450 (0,43113; 0,47924) 1 42 0,50464 0,05823 (0,48146; 0,52782) 3 41 0,5215 0,0779 (0,4981; 0,5450) 4 84 0,65731 0,09037 (0,64092; 0,67370) Pooled StDev = 0,0761889

Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

Type N Mean Grouping

4 84 0,65731 A    

3 41 0,5215   B  

1 42 0,50464   B  

0 39 0,45518     C

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Figure 7 test to see which groups were significant from each other in HM:EM ratio found in type 0,1,3 and 4 chickens. Type 0, No IP. Type 1, IP. Type 3, EP. Type 4, Hatchlings. Data points were presented as means.

PRIMARY WORKSHEET

One-way ANOVA: LM:EM versus Type

Method

Null hypothesis All means are equal

Alternative hypothesis Not

all means are equal

Significance level α = 0,05

Equal variances were assumed for the analysis. Factor Information

Factor Levels Values

Type 4 0; 1; 3; 4

Analysis of Variance

Source DF Adj SS Adj MS F-Value P-Value

Type 3 36,90 12,2990 64,22 0,000 Error 202 38,69 0,1915     Total 205 75,58       Model Summary S R-sq R-sq(adj) R-sq(pred) 0,437634 48,82% 48,06% 47,12% Means

Type N Mean StDev 95% CI

0 39 2,4990 0,3103 (2,3608; 2,6371) 1 42 2,5714 0,3460 (2,4383; 2,7046) 3 41 2,5138 0,3239 (2,3790; 2,6485) 4 84 3,3887 0,5589 (3,2945; 3,4829) Pooled StDev = 0,437634

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25 Tukey Pairwise Comparisons

Grouping Information Using the Tukey Method and 95% Confidence

Type N Mean Grouping

4 84 3,3887 A  

1 42 2,5714   B

3 41 2,5138   B

0 39 2,4990   B

Means that do not share a letter are significantly different.

Figure 8 test to see which groups were significant from each other in LM:EM ratio found in type 0,1,3 and 4 chickens. Type 0, No IP. Type 1, IP. Type 3, EP. Type 4, Hatchlings. Data points were presented as means.

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Figure 8 & 11 Anova and tukey test on absolute heart mass light group (group 1) from table 2.

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Figure 9 & 12 Anova and tukey test on absolut heart mass middle group (group 2) from table 2

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Figure 10 & 14 Anova and tukey test on absolute heart

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Significant increase in liver and heart mass found in post hatching red junglefowls (Gallus gallus).