The Effect of Heat Stress on the Livers of Laying Hens vs Non Laying Hens Presentation

The Effect of Heat Stress on the Livers of Laying Hens vs Non Laying Hens Presentation


The effect of heat stress on the livers of laying hens vs non-laying hens (Mariane Houle) Mariane Houle (Micah Ang) and I’m Micah Ang (Mariane Houle) and our project is the effect of heat stress on the livers of laying hens versus non laying hens. (Micah Ang) So we’re actually
a sister team of the team eggs that you heard earlier, and our central research
question that we’ve answered over the three months that we had this summer
goes, what is the effect of heat stress on the liver of adult hens. (Mariane Houle) So just to
give you some background about eggs and why they’re important. So the global
population of the world is supposed to increase from 7.6 billion to 9.8 billion by 2050. And that means we need more food to feed these people. So
chicken eggs are actually a major source of protein and nutrients for people in
developing countries and commercially about ten percent of the eggs produced are
soft-shelled which is a bad thing because it’s a negative financial loss
for farmers. And that means that there’s less food in the world for us to eat. And
in our project we’ll be looking at the liver of chickens. And the liver is a
vital organ because it has many functions. It metabolizes things it
detoxifies things. And it’s also involved in red blood cell formation in birds. (Micah Ang) Alright, and so here we have the major stresses or the problems that we had to
deal with with our research projects. So first off we have heat stress. This is
actually one of the main culprits in the decline of chicken health and even in
the egg marketability and that’s why we want to focus on it. So by definition
heat stress occurs when high environmental temperature increases the
core body temperature of an organism above its normal condition – so above its
optimal working conditions at which an organism or in this case the organ
functions. So that is the macro problem that we delve into. And then the second one is we’re going down to the molecular level.
We have oxidative stress which is caused by the heat stress. Basically oxidative
stress is an imbalance between the presence of free radicals which is not
good for the organism’s body, and there is again an imbalance between that and the
presence of antioxidants. So in general if there are more free radicals than
antioxidants then you are under oxidative stress. (Mariane Houle) So what is a free
radical and why is it bad? So if you remember like way back to gen chem, a free
radical is basically an atom that lacks an electron so in order for it to
become stabilized it’ll steal an electron from a healthy atom and that’s
bad because if you have too many free radicals in your body it’ll lead to DNA
damage, protein impairment, and cellular function impairment. And antioxidants
basically donate an electron to the free radicals and that helps to stabilize it.
So that’s why you want more antioxidants in your body in order to mitigate the effects of free radicals. (Micah Ang) And so, so as we mentioned earlier, our focus is on the
organ, the liver. Heat stress has actually negative physiological effects on this
organ, massively, because we know that the liver is the bloodline of the body.
It does a lot. It’s a 24/7 ongoing working machine. And due to heat stress
it actually impairs the endocrine system and even though reproductive functions
of the chicken. And we know that when the liver is under heat stress the liver tends to double time its work in regaining
homeostasis and just maintaining its functions for the body’s overall health. (Mariane Houle) So Dr. Mishra’s team at CTAHR at UH Manoa actually conducted a trial where they
placed hens under the following conditions for three weeks. So the non
heat stress hens were exposed to temperatures ranging from 21 to 22
degrees Celsius at 50% relative humidity and the heat stress chickens were placed
at 30 to 35 degrees Celsius temperatures from 42 to 50 percent relative humidity.
And they found that the livers of the heat stress chickens were actually
extremely fragile. And this is possibly due to the fact that they were being
overworked. And basically what that means is if you have a bad liver then the
chickens have bad egg quality. (Micah Ang) And so next up this
is one of the major segments of our research project. We begin with this gene
called TATA binding protein. It’s a housekeeping gene that is present among
all organisms and it encodes for a TATA box, a binding protein. So prior to
monitoring six major genes of interest in the liver tissue we use this gene as
a baseline. So basically, it it provides a an equal playing field for all the 35
samples that we have for our research project. And so, since again, it’s present amongst
all organisms, it’s a good candidate to be a baseline or form of comparison. So
here are six major genes of interest. All throughout the three months that we had we monitored them either being upregulated or downregulated. And as you
can see we divided them into three categories. We have first one it’s enzyme
related. The second one is cell maintenance related, and then the last
one is protein related. So here in the first column we have these three genes
first off we have ROS1 ROS1 encodes for an enzyme that is
responsible for cell growth, cell differentiation, and cell signaling. And
ROS actually also stands for reactive oxidative species which is very which is
a key word since we’re dealing with oxidative stress. And in the second one
is CAT. It encodes for the enzyme called catalase. It’s an antioxidant that
scavenges free radicals such as the hydrogen peroxide and breaks it down to
a viable molecule such as water. And in the last one is the SOD1. It encodes
for the enzyme superoxide dismutase, and it breaks down superoxide free radicals. (Mariane Houle) So
the last genes that last three genes that were looking at are basically
involved in cell maintenance and protein production. CASP3 is involved in
apoptosis, HSPA9 basically encodes for heat shock protein, and HSP90AA1 encodes
for a protein that aids in proper protein folding. And basically we’re
looking at the “eggs-pression” of these genes so, Glad you got that thanks Dr. Kae. Alright. So based on what we know of
these genes of interest we believe that heat stress will up
regulate the expression of genes such as ROS1 CAT and SOD1 in the liver of
heat stress versus the non heat stress hens. (Mariane Houle) So we believe CASP3 HSPA9 and HSP90AA1 will be downregulated in gene expression in the liver of heat stress
versus non heat stress hens. So for our methods. (Micah Ang) Alright. So down to the
nitty-gritty part of our experiment. We actually partnered up with a research
team at CTAHR at UH Manoa and our research mentor was Dr. Birendra Mishra.
And they did the dirty work for us. So they they took the they collected the
samples of the chicken liver tissues. We did not kill the chickens. And so from
that tissue we extracted the total RNA from the tissue and we we used that we
isolated that and used that as a building block for the synthesis of the complementary DNA that will be a template for the next method. (Mariane Houle) So the next
method that we did mainly here at LCC was qPCR which is quantitative pcr. It
basically measures the concentration of DNA over time and that can help us
determine the levels of gene expression. And we also did gel electrophoresis just
to confirm our qPCR results. So for our results section this graph shows you
the gene expression levels of ROS1 CAT and SOD1 which are the oxidative
stress genes. We’ll mostly be focusing on ROS1 because that’s the only gene
that we found showed a significant difference between all of our samples
and we’ll explain that later. This graph also shows the gene expression
levels of CASP3, HSPA9, and HSP90AA1 and that’s not really relevant
because it didn’t really show a significant difference between samples
as well. (Micah Ang) Right. And just to note right here the y-axis is the Fold Induction. So if you see that the bars are higher meaning that there is more
gene expression. But since we only found out that there is a significant
difference amongst the samples in the gene ROS1 we are only going to focus
on this gene. But for the record we did run a statistical test on all of the
genes. And the statistical test is called one-way ANOVA. ANOVA stands for analysis
of variance and and so with the help of Excel we were able to do the necessary
computations and we came up with these values. So we have here the F statistic,
the critical value, p value, alpha level. These numbers are
going to be very important in to how we interpret the data. And so here if you
can see here in the first bullet on the left- on your right side, if the F
critical value is less than the F statistic, bear with me I’m gonna be
doing a tiny statistic lesson, but so if you have that a key word is less than
then we can say for sure that there is a statistical difference or we can say
that the gene is upregulated compared to another. And then the second
way of interpreting the the data that we had was comparing the p-value and the
Alpha level. Again when the p-value is less than the Alpha level which in case
in our case did check off, then we can definitely
conclude that there is a significant difference in at least two pairs amongst
the five groups of hens that we have. But then this test is not enough because we
don’t know where the difference lies, and so there’s another test. (Mariane Houle) So this test is
called the post hoc Tukey Kramer test. And that basically tells you where the
difference is in between groups, if you have more than two groups, which we have
five. So we basically look at our our Q critical value which we got
off of a table. So that was a given value. And then if the Q critical value is less
than the Q statistic which we calculated, then there is a significant difference
between two groups. And we found that there was a significant difference
between the non laying hens and the heat stress hens as well as the molter hens
and the heat stress hens. So this is the our gel results from ROS1 and we’re
just showing you this because we ran a gel for all of our genes but in the
interest of time we’re only showing this one. So this just shows you that all of
our bands did fall within the expected base pair range. So our base pair range
was 225 and this is 200 base pairs. This is 300 base pairs. So everything pretty much
looks good this just confirms that our primaries did work and that everything is going as planned. (Micah Ang) And we did run a gel on each of the genes and it all checked off. And in conclusion we found out that ROS1 gene was the only gene to show a significant difference in expression
levels between the heat stress and the non heat stress levels
I mean heat stress hens. And mentioning that (Mariane Houle) So this increased expression of
ROS1 which again is the reactive oxidative species can be attributed to
the fact that there was an increase in oxidative stress between these hens
specifically the heat stress hens and it’s bad because it does oxidative
stress does alter metabolism and it decreases egg quality, yeah. (Micah Ang) And um if if
you remember the graph that we had previously you could you could really
see the staggering difference. That there was an upregulation within the heat
stress groups when it comes to the gene ROS1. And it’s really bad because again
Mariane told that it alters metabolism it debilitates the egg-laying productivity
of the chickens. And the last one we have – so within the ROS gene we found out
through the statistical test that we did, that across the 5 samples that we have
there was a significant difference between the non laying and the heat stress
and then the molters versus the heat stress, but none showed between the layers
versus the heat stress. (Mariane Houle) So references and we’d like to thank Dr. Kae Dr. Neupane for their unconditional support we appreciate it,
and our lab mentor at Manoa Dr. Mishra and his team as well as the funding for
this program. So thank you so much we appreciate it. Thank you (Micah Ang) Any questions? Any questions at all? (Mariane Houle) No? Good. Okay, let’s go. Thank you.

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