Topic title

Pretentious genomic selection signatures in CYP19A1 gene

associated with silent estrous behavior in water buffalo in Pakistan

Student Name: ZARA SALEEM

Course Instructor: Nighat Syed

 Self Introduction

Student Name: ZARA SALEEM

Student ID: BC180400939

Study Program: BS Biotechnology

Course name: Seminar II

Course Code: BT602

Current semester no: 8th

 1 Introduction of topic

2 Materials and methods

3 Results
Contents
4 Discussions

5 Conclusions

6 References
 1. Introduction of Topic
 Buffalo are considered as very important animals for agricultural economy.

They provide us milk, meat and fur etc. Their milk is chosen mostly
because of high fat and protein. Buffalo contributes about 68% to the total
milk production in our country (Bilal et al., 2006).

 Buffalo are like shy breeders as they face some reproductive constraints.

Silent heat or silent estrous is one of that constraints(Imran et al., 2018).

 Estrous is a phase in which females become sexually accessible, and estrous

cycle is a time period from one estrous to a following estrous.

 It is a time period in organisms beginning with one ovulation to a
subsequent ovulation. This cycle contains changes which are induced by
reproductive hormones. (Thakur et al., 2013).

 Estrous is a phase of sexual receptivity in animals. High amount of Estrogen

hormone is present and this hormone is responsible for behavioral symptoms

of estrus. Detection of estrous is important for timely insemination.

 Estrous or heat signs are mucus discharge, swelling and redness of vulva,

sniffing, mounting, licking, back rubbing, less feed intake, decreased milk
production, aggression, increased urination, vaginal discharge and bellowing
(Imran et al., 2018).
 Silent estrous or silent heat is a condition in which buffalo does not show

behavioral symptoms of estrous. During this, physiological signs of estrous

are present. During silent heat, animals fail to express only behavioral
symptoms of estrous while ovarian changes and hormone secretions occur
normally.

 Due to silent heat, breeders are unable to detect heat and it causes problems

in timely inseminations. So silent estrous is a major cause of less

exploitation of production potential of these useful animals(Imran et al.,
2014, Imran et al 2018).

 By studying the genes involved in expression of estrous and its mechanism,

we can get information about reasons of silent estrous.

 To study the reasons behind silent heat and estrous expression, this current

study was focused on CYP19A1 gene and SNPs in this gene because this
gene has close association with silent heat (Kommadath et al., 2011).

 In bovine species, CYP19A1 gene is present on chromosome 10 and has 10

exons. This gene codes for aromatase protein. It was mapped to q2.6 band.
The size of this gene ranges form 56 to 120 kbs in different species (Cho et
al., 2012).

 In this study, CYP19A1 gene was characterized to identify different genomic

variants that have close association with silent estrous behavior in buffalo.
PCR amplification and sequencing techniques were used to find and analyze
these genomic variants.
 2. Material and methods

In this study, we are going to discuss and find SNPs present in CYP19A1
gene that has effects on estrous behavior of Nilli Ravi buffalo. These SNPs
results in poor estrous expression.

2.1. Animal selection criteria:

A total of 97 animals were selected having typical phenotypic features. These

animals were genetically related but belongs to different families. These
animals belong to different breeds and have fertility record up to 3
reproductive cycles.
2.2. Sampling method:

Animals are categorized in two groups on the basis of estrous efficiency, one
group whose all members show good heat signs while other group’s members
showing poor heat signs by a method reported by (Reolofs et al., 2005).

In this method, behavioral signs of heat were recorded after 2 or 3 intervals.

Score was assigned to each sign. At the end, if the total sum of score of any
animal is equal to 50 during two successive observations, then it means that
animal shows good heat. If total sum of score doesn't exceed 50, then it means
that animal is not showing good heat signs.
On the basis of these observations, animals are characterized into 2 groups.
One group that showed good heat signs while other that showed low heat sign.

Season is important parameter to be kept in view while observing heat signs in

buffalo because buffalo shows good estrous signs in autumn season.

2.3. Blood sampling:

Blood sampling was carried out for both groups from jugular vain. In this step,
blood samples from each animal is collected and transferred to a 50 ml Falcon
tube having 200 micro liters of ADTA (an anticoagulant). After collection of
blood samples, these samples were placed on ice and stored at -20 °C until
DNA extraction.
2.4. DNA extraction and quantification
Inorganic method of DNA extraction was used to extract DNA from blood
samples(Maryam et al., 2011, Sambrook and Russell, 2001). Nanodrop and
gel electrophoresis are used for DNA quantification.

2.5. Polymerase Chain Reaction

To perform PCR, primer designed from the coding regions of CYP19A1

gene(exon no 2,3 and 9) were used. Touchdown PCR, subsequent PCR,
forward and reverse primers and water were used. After 30 cycles, the product
having DNA was separated with the help of gel electrophoresis.
2.6. Gel elusion and sequencing

After the amplification of DNA, FavorPrep GEL/PCR Purification kit was used for
elusion purpose. To observe its equality, it is loaded onto 0.2% gel. Sanger’s Chain
termination method was used for sequencing of PCR amplicons after elusion.

2.7. Statistical and bioinformatics analysis

The CHROMAS software were used to analyze sequences. The ClustalW software
was used to align sequences of both groups. After that, total 11 SNPs were
recognized. The POPGene.32 software was used to perform population genetic
analysis. To check the level of significance of each loci, the chi2 test for HWE was
performed. Association analysis was performed for those loci which obeyed HWE.
Phyre 2 tool was used to determine three dimensional protein structure of
CYP19A1 gene.
 3. Results

3.1. Single marker analysis

We found total 11 SNPs and among these, total 7 SNPs were found in coding
region while remaining four were in non coding regions. This analysis
provided allelic frequency of every polymorphic site. For more of the
polymorphic sites, CYP19A1 gene was found homozygous and very less
heterozygosity was found. Only five SNPs out of 11 showed strong association
with poor heat score in buffalo.
3.2. Single nucleotide polymorphism
As we discussed earlier, we find total 11 SNPs and among these, total 7
SNPs were in coding region while remaining 4 were in non coding region.
Only in exons 2 and 9, four amino acid substitutions were observed.
Only one SNP present in exon 9 of gene, which changes the effect of AA
substitution from non-polar to polar as given below in table no 1.
Table no. 2
Identified novel polymorphic sites in exons 2 and 9 in the CYP19A1 gene.

SNPs Location Wild Mutant Transition/ Amino acid Effect

Variation variation Transversion substitution

p.I23V Exon 2 A G Transition Isoleucine> Non-

Valine polar

p.V369D Exon 9 T A Trans version Valine> Non-

Aspartate polar>
polar
3.3. Three dimensional structure of protein
To check the location of exonic polymorphism, a three dimensional
protein’s structure were established.TheCYP19A1 protein carries various
functionally significant regions . The SNP p.V269D was found in the
steroid binding domain, disposing the protein activity toward the more
polar side (fig. 1). The SNP p.I28V was located as a part of the
transmembrane helix of the protein in the mitochondrial membrane.
These mutations describe the variation in protein binding and activity,
showing that the aromatase sequence has been altered because of these
mutations, thereby causing silent estrus behavior.
Fig. 1. Three-dimensional predicted protein model of
CYP19A1 by homology modeling (Imran et al., 2018).
Fig. 2. Structural configuration of the CYP19A1 gene and the identified
polymorphic sites potentially associated with silent heat in Nili-Ravi buffalo
(Imran et al.,2018).
 4. Discussions

The role of CYP19A1 gene in regulation of reproductive hormones and

to identify polymorphic sites had been studied (Sambrook and Russell.,
2001, Van et al., 1996). It clearly depicts the polymorphic nature of the
gene, especially its tendency towards estrous cyclicity.

Suneel et al., (2009) reported 4 SNPs in Murrah Buffalo. SNP identified

in the exon 2 region of the CYP19A1 gene, which was a transversion,
was reported by Suneel as compared to the SNP identified in the exon 2
region of the CYP19A1gene in the current research.

SNPs in the CYP19A1 gene have also been reported by Cochran et al.,
2013. They studied the role of CYP19A1 in controlling the silent estrus
behavior in hostlein cow.
SNPs in the Bovine CYP19A1 gene have been reported by Vanselow et al.,
2000. The SNPs identified by Vanselow et al. were both transitions.
SNPs in the CYP19A1 gene have also been reported by Cho et al., 2012.
These authors demonstrated that these mutations cause an increase or
decrease in the aromatase activity, thereby altering the levels of the
circulating estrogen.
In the past, several improvements have been made in dairy animals like
cow and buffalo to increase milk production which affect fertility rate of
these animals. There is a negative association in milk trait and fertility in
cow and buffalo. So the reproductive capabilities remain unexploited
because of these improvements and reproductive constraints like silent
estrous.
In current research, those SNPs which we found are novel for Nili-Ravi
buffalo because they were not previously reported in any study. In this
research, SNPs present in intronic regions didn’t have any effect on
binding site, splice junctions and transcript machinery. But it can cause
gene silencing through interference techniques.

Due to high polymorphic nature of CYP19A1 gene, it can be a pretentious

candidate to identify selection signature. These genomic signatures can
prove useful in future for breeding programs to produce animals with
superior reproductive efficiency.
 5. Conclusions

From all these results and discussions, it can be concluded that

identified SNPs in this current research can be used as genetic markers

for silent estrous behavior. Silent estrous is a major reproductive

problem in Nili Ravi buffalo. These genetic markers could proved very

useful in genetic evolution and selection of breeds with superior

efficiency.
 6. References
Primary reference:
 Imran, S., Javed, M., Nadeem, A ., Iqbal,M. (2018). Pretentious genomic
selection Signatures in CYP19A1 gene associated with silent estrous behavior
in water buffalo of Pakistan. Electron J Biotechnology 32: 35-40,
https://doi.org/10.1016/j.ejbt.2018.01.001

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