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Sex determination of ovine embryos by SRY and amelogenin (AMEL) genes using maternal circulating cell free DNA ANIMREP 2016 (164) 9-13

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Animal Reproduction Science 164 (2016) 9–13
Contents lists available at ScienceDirect
Animal Reproduction Science
journal homepage: www.elsevier.com/locate/anireprosci
Sex determination of ovine embryos by SRY and amelogenin
(AMEL) genes using maternal circulating cell free DNA
Adel Saberivand a,∗ , Sima Ahsan b
a
b
Division of Theriogenology, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran
Graduate in Veterinary Medicine, Urmia University, Urmia, Iran
a r t i c l e
i n f o
Article history:
Received 27 December 2014
Received in revised form
23 September 2015
Accepted 30 October 2015
Available online 1 November 2015
Keywords:
Sex determination
Amelogenin
SRY
Embryos
Ghezel Sheep
PCR
a b s t r a c t
Simple and precise methods for sex determination in animals are a pre-requisite for a
number of applications in animal production and forensics. Some of the existing methods
depend only on the detection of Y-chromosome specific sequences. However, the detection of Y and X-chromosome specific sequences is advantageous. In the present study the
accuracy of sex determination by SRY (sex-determining region Y) and AMEL (Amelogenin)
gene detection was assessed using a polymerase chain reaction (PCR) of DNA extracted
from free fetal cells in maternal blood, which is noninvasive for fetus and easier to collect.
The PCR amplification of SRY primers produced a single band of 171 bp from ewes bearing
a male fetus, whereas no band was amplified from the DNA extracted from ewes pregnant
to a female fetus. Moreover, two bands of 182 and 242 bp in male and a single band of
242 in female fetuses were produced by AMEL gene primers in the PCR reaction. Using
this technique 100% of samples were successfully sexed, excluding twins. In conclusion, we
demonstrated that sex determination using DNA of free fetal cells in maternal plasma is
efficient using both SRY and AMEL gene sequences. It also is evident that this method is not
suitable for sex determination of twin pregnancies.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
One of the basic and important elements of animal production is scientific progress on reproductive technologies
(Dervishi et al., 2011). On the other hand, fetal sex detection
is a key practice in livestock management. It is extremely
important in ovine industry to identify lamb gender for
breeding, culling and eliminating expenses of the progeny
test programs (Kadivar et al., 2013).
∗ Corresponding author at: Division of Theriogenology, Department of
Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, 29
Bahman Blvd., Tabriz, Iran.
E-mail addresses: [email protected], [email protected]
(A. Saberivand).
http://dx.doi.org/10.1016/j.anireprosci.2015.10.011
0378-4320/© 2015 Elsevier B.V. All rights reserved.
Several different methods have been used to determine
mammalian prenatal sex. These methods are: trans-rectal
ultrasonography (de Freitas Neto et al., 2010), cytogenetic
analysis (King, 1984), detection of H-Y antigen (Watchel,
1984), measurement of X-linked enzymes before Barr body
formation (Kageyama et al., 2004). The need to harvest cells
in metaphase is a limitation of cytological detection. In situ
hybridization and fluorescent in situ hybridization (FISH)
is time consuming (Dervishi et al., 2011) and the success
of trans-rectal ultrasonography is depended on the fetal
position, age and the operator’s experience (Ali, 2004).
Although the presence of fetal nucleic acids in the
maternal circulation was discovered decades ago (Mandel
and Métais, 1948), circulating cell free fetal DNA (ccffDNA),
was just identified in maternal plasma in late 1990s (Lo
et al., 1997) when they correctly identified male fetuses
in 80% of the pregnant women carrying male fetus. This
10
A. Saberivand, S. Ahsan / Animal Reproduction Science 164 (2016) 9–13
revolutionary-like event has emphasized that the placenta
is no longer thought to be an impermeable membrane
(Swarup and Rajeswari, 2007) and provided new approach
for prenatal molecular diagnosis such as fetal sex determination, screening for pregnancy-related complications and
fetal diseases (Lo et al., 1998; Jimenez and Tarantal, 2003;
Maron et al., 2007).
Embryonic sex determination using sex-specific DNA
sequences by Polymerase chain reaction (PCR) has been
used in cattle (Peura et al., 1991; da Cruz et al., 2012),
pigs (Fajfar-Whetstone et al., 1993), humans (Handyside
et al., 1989) and mice (Kunieda et al., 1992). Because of
acceptable reliability, high sensitivity, inexpensiveness and
rapidity, PCR is currently used as genotypic sex determination method (Dervishi et al., 2011).
The main drawback of this method is that the absence
of amplification is interpreted as the fetus being female. To
overcome this issue, a gene which is present in male and
female should be amplified at the same time. Amplification
of ZFX/Y region and Restriction Fragment Length Polymorphism analysis (Aasen and Medrano, 1990) and Duplex PCR
co-amplifying a specific Y-chromosomal sequence and an
autosomal sequence as a control have been used (Appao
Rat and Totey, 1999; Mara et al., 2007).
Another issue is the type of ovine placenta. Unlike the
humans (hemochorial), sheep has a synepitheliochorial
placenta which does not permit a direct contact between
the maternal blood and fetal trophoblast (Wooding, 1992).
Therefore, the likelihood of the passage of fetal DNA to
maternal circulation is very scarce, highlighting the importance of the sensitivity of DNA extraction methods to be
used.
It is the presence or absence of the Y chromosome
determines the sex of fetus during sexual differentiation.
Therefore, the sex determining region, SRY has been the
most frequent gene used for sex typing (Piprek, 2010),
and seems to have greatest homology within the ruminant
species (Pomp et al., 1995).
The amelogenin (AMEL) gene, which exists on both X
(AMELX) and Y (AMELY) chromosomes, has been used to
determine the sex in cattle (Ennis and Gallager, 1994),
sheep and deer (Pfeiffer and Brenig, 2005; Dervishi et al.,
2011; Kadivar et al., 2013).
The aim of the present study was to use cell free fetal
DNA in pregnant ewe plasma to determine fetal sex in a PCR
assay using SRY and amelogenin (AMELX/AMELY) genes.
2. Material and methods
2.1. Blood collection and plasma harvesting
Fifty one mature Ghezel ewes in gestational weeks of
8–20 from private sector and from animals brought to
government-owned clinics were used in this study.
In addition, three normal non-pregnant ewes and three
normal rams were used as control animals. As a source
of ccffDNA, peripheral blood samples were obtained from
the animals. A volume of 5 mL of blood samples was collected from jugular vein into the EDTA vacutainer tubes
and centrifuged at 1600 × g at room temperature for 10 min
to separate plasma from packed cells and buffy coat.
Subsequently, they were centrifuged at 16,000 × g for
10 min to further separate cellular debris. The blood plasma
samples were stored at −20 ◦ C until analysis.
2.2. DNA extraction from maternal blood plasma
DNA was extracted from 100 ␮l plasma using a commercial DNA Purification kit (Sinaclon, Iran) as recommended
by the manufacturer. The total DNA extracted from the
cells was used as template in PCR for sex determination.
The quality and quantity of DNA were determined using
Biophotometer (Eppendorf, Germany).
The primers were synthesized (Sinagen, Iran) for SRY
and AMEL according to Dervishi et al. (2011) and were as
follows; SRY gene; upstream: 5 - GACAATCATAGCGCAAACGA-3 , downstream: 5 -CAGCTGCTTGCTGATCTCTG-3 ;
AMEL gene; upstream: 5 -CCGCCCAGCAGCCCTTCC-3 ,
downstream: 5 -CCCGCTTGGTCTTGTCTGTTGC-3 .
Amplification conditions were identical for two genes
except the thermal profile as is stated. The amplification
reactions were set in a final volume of 25 ␮l containing
5 pmol of each primer, 200 mM of each dNTPs, 2 mM MgCL2 ,
50 mM KCL, 10 mM Tris–HCL, 0.5 U Taq DNA Polymerase
and 100 ng of genomic DNA.
The thermal profile was as follows; initial denaturation
at 94 ◦ C for 5 min. Thirty-five cycles with the following
step-cycle profile: denaturation at 94 ◦ C for 45 s, followed
by primer annealing at 63 ◦ C (for AMEL) and 55 ◦ C (for SRY)
for 45 s, and primer extension at 72 ◦ C for 45 s. The last
extension step was 10 min longer. An aliquot of each reaction mixture was subjected to electrophoresis in 2% agarose
gel and stained with safe stain (Sinaclone, Iran). The PCRbased sex of embryos were compared with the phenotypic
sex and presented as percentage data. Efficiency of the
method was evaluated in percentage (%) for both genes.
3. Results
Fetal male (SRY and AMELY) and female (AMELX) circulating DNA was successfully indicated in the maternal
plasma in the gravid ewes. Out of 51 samples, 8 samples
(15.68%) were twins that were not used in the analysis.
For comparison, one sample of twins was used and analyzed (data are not included). The stage of pregnancy and
PCR detected sex by PCR for both SRY and AMEL genes are
summarized in Table 1. There was no significant difference
between stages of pregnancy for both genes determining
fetal sex.
The PCR amplification of SRY primers on DNA extracted
from blood plasma of ewes bearing a male fetus produced
a single band of 171 bp, whereas no band was amplified
from the DNA extracted from ewes pregnant to a female
fetus (Fig. 1).
Twenty one out of 23 (91.30%) female fetuses were
correctly identified by SRY gene sex determination. The
number of male fetuses identified by this gene was 19 out
of 20 (95%). The overall test accuracy for correct sex determination for SRY gene was 93.15%.
Two bands of 182 and 242 bp in male and a single band of
242 in female fetuses were produced by AMEL gene primers
in the PCR reaction (Fig. 2). Twenty two out of 23 (95.65%)
A. Saberivand, S. Ahsan / Animal Reproduction Science 164 (2016) 9–13
11
Fig. 1. PCR products of SRY gene from pregnant ewe plasma samples. Lane 1: negative control, lane 2: positive control, lanes 4, 5 and 6 ewes with male
fetuses. Lanes 3 and 6, ewes with female fetuses. Lane 8, 100 bp DNA marker.
Fig. 2. PCR products of AMEL gene from pregnant ewe plasma samples. Lane 1: 100 bp DNA marker. negative control, lane 2: positive control female ewe,
lane 3: positive control male ewe, lanes 4 and 7 ewes with female fetuses. Lanes 5, 6 and 8, ewes with male fetuses.
female fetuses were correctly identified by AMEL gene sex
determination. The number of male fetuses identified by
this gene was 19 out of 20 (95%). The ovine AMEL gene PCR
for sex determination had a sensitivity and specificity of
97.82% in this study.
Out of 45 samples used in this study, 8 samples were
twin pregnancies. One ewe carrying two female fetuses
showed a PCR pattern like single female fetus and the ewe
bearing twins with both sexes indicated a male-like PCR
pattern. Excluding the twin pregnancies, the efficiency of
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A. Saberivand, S. Ahsan / Animal Reproduction Science 164 (2016) 9–13
Table 1
Ovine fetal sex determination by PCR using both SRY and AMEL gene
primers from free fetal DNA of maternal blood plasma in 45 pregnant
ewes.
Pregnancy
trimester
Phenotypic
sex
PCR detected
sex by SRY
gene primers
PCR detected
sex by AMEL
gene primers
Specimen
no.
1
3
1
1
2
2
3
1
2
1
3
1
2
3
1
2
3
1
1
1
3
3
3
2
1
1
3
2
2
1
2
1
2
3
2
3
2
3
2
3
2
3
3
M
F
M
M
F
F
M
F
M
M
M
M
F
F
M
F
F
M
F
M
F
F
M
M
F
F
F
F
M
F
F
M
M
M
F
F
F
F
M
F
F
M
M
M
F
M
M
M
F
M
F
M
M
M
F
F
F
M
F
F
M
F
M
F
F
M
M
F
M
F
F
M
F
F
M
M
M
F
F
F
F
M
F
F
M
M
M
F
M
M
F
F
M
F
M
M
M
M
F
F
M
F
F
M
F
M
F
F
M
M
M
F
F
F
M
F
F
M
M
F
F
F
F
F
M
F
F
M
M
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
using both genes in a PCR reaction to determine fetal sex
on the maternal plasma was 95.48%.
4. Discussion
This is the first report of ovine fetal sex determination
using both SRY and AMEL genes in maternal circulating
cell free DNA in Ghezel breed. The PCR reaction using SRY
primers only produced a single band of 171 bp in male pregnancies. While, AMEL primers amplified two bands of 182
and 242 bp in male and a single band of 242 bp in female
pregnancies.
Maternal circulating cell free DNA was previously used
for fetal sex determination and to detect fetal diseases and
abnormalities in mammals (Bianchi and Lo, 2001; Guang
et al., 2009; Davoudi et al., 2011; Kadivar et al., 2013).
Kadivar et al. (2013) identified fetal sex with the sensitivity
and specificity of 100% with no false negative or false positive results using SRY gene in plasma samples of pregnant
ewes. Similarly, ovine fetal sex has correctly been identified
in 579 out of 580 samples using SRY gene (Dervishi et al.,
2011). In another research (Vanpé et al. (2013) both SRY
and AMEL genes were used to successfully identify fetal
sex in 3 lemur species.
Reliable ovine fetal sex determination could be a key
element in ovine management and breeding programs. The
traditional methods of fetal sex determination are uncertain and time-consuming and most of them are invasive
and have potential risk of pregnancy damage. PCR using
the sequence differences between Y and X chromosomes
in male and female sex provides precise fetal sex determination. Successful isolation and quantification of cell free
fetal DNA from maternal plasma is dependent on plasma
harvesting from whole blood samples, DNA extraction from
harvested plasma; and PCR quantification or detection of a
paternally inherited sequence (Maron et al., 2007).
Often the quantity of samples such as hair, urine, skin
or fetal cells in maternal circulation for DNA extraction is
very low. Therefore, it is important, especially in forensics
and crime investigations, to use methods and tools that are
able to extract DNA from such a small amount of samples
and, to detect the specific sequences in the small quantity
of samples (Vanpé et al., 2013). In wild animals, especially
where they are in the risk of extinction, safe fetal sex determination using molecular methods without compromising
fetal life is crucial (Waits and Paetkau, 2005).
Application of both SRY and AMEL genes in maternal
circulation may be useful where female embryo’s genotype is XY and due to lack of SRY gene sequence its sex is
incorrectly determined as female. There are some reported
evidences in humans that infertile males with XX genotype
have determined as female using Y chromosome-specific
variants (Rajender et al., 2006).
DNA contamination with human origin some time could
be a problem in sex determination with SRY-specific variants. Therefore, using AMEL gene can overcome this issue
as human AMEL gene primer produces different product
size of animal AMEL gene (Pfeiffer and Brenig, 2005).
The main drawback of this method is the failure to
detect the gender of twins. As Ghezel has a twining rate
of 20% (Mohammadi and Saberivand, 2012) and there are
many ovine breeds that have very high twining rate, this
method is not able to distinguish between female fetus and
mother AMEL-X product. More investigations are required
to develop methods capable of identifying DNA of female
fetus from maternal DNA. Multiplex PCR including both
genes primers would have been very accurate method if
the annealing temperature of the primers were set to be
very similar. Quantitation of a PCR product from a plasma
sample of pregnant ewe against a verified DNA sample from
the same dam in the quantitative real-time PCR using AMEL
gene primers might be useful.
Acknowledgments
This work was supported by funds granted by the Vice
Chancellor for Research of Urmia University. The authors
A. Saberivand, S. Ahsan / Animal Reproduction Science 164 (2016) 9–13
thank Dr. A. Ghadami and Dr. P. Aparnak for their collaboration in sampling and data collection.
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