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POLYMERASE CHAIN REACTION AND FLUORESCENT IN SITU

HYBRIDIZATION USED FOR BOVINE EMBRYO SEXING

Assistant Mihai CENARIU*, PhD

Professor Ioan GROZA*, PhD

Professor Liviu BOGDAN*, PhD

Emoke PALL*, PhD Student

Lecturer Simona CIUPE*, PhD

Abstract

The purpose of this paper was to evaluate the results obtained for bovine embryo sexing using the polymerase chain reaction (PCR) 
and fluorescent in situ hybridization (FISH) and to compare them in order to decide which of the two methods is more accurate and 
yields better results while necessitating less effort. We took into consideration the pregnancy rate obtained after the transfer of biopsied 
embryos, the percentage of correctly sexed embryos evaluated at birth (when the predicted sex was compared with the actual sex of 
the newborn) as well as other characteristics related to the difficulty of the method, expenses and suitability to a minimally equipped 
laboratory. We concluded that the polymerase chain reaction is the most accurate and suitable method for sexing preimplantation 
bovine embryos, being in the same time easier to perform than the fluorescence in situ hybridization.

Key words: bovine embryo, biopsy, polymerase chain reaction, fluorescent in situ hybridization

Introduction

Preselection of the sex of offspring of agriculturally important species has long been an objective 

of animal breeders (Bredbacka, 2001, Groza et al., 2006, Cenariu et al., 2008). However, not until the 

advent of technologies such as artificial insemination and embryo transfer has such an approach been 

considered commercially feasible (Cenariu et al. 2008, Groza et al., 2005). Preliminary studies suggest 

that methods for sexing embryos do show potential for commercial use (Groza et al., 2006, Lee et 

al., 2004, Taketo et al., 2005). Embryo sexing will be used in conjunction with embryo transfer and 

most of the research has been geared to sexing bovine and equine embryos, which can be transferred 

non-surgically up to the blastocyst stage (Jin and Lloyd, 1997). Sexing methods are classified as 

either invasive or noninvasive, depending on whether or not a biopsy of embryonic tissue is required 

(Shea, 1999, Cenariu et al., 2008). Criteria that must be considered in embryo sexing techniques are 

* University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Department of Veterinary Reproduction, 
Obstetrics and Gynecology, 3-5 Manastur Street, 400372 Cluj-Napoca, e-mail: mcenariu@yahoo.es

Cluj Veterinary Journal, 15(1)/2009, pp. 19-23



20

the percentage of embryos that can be accurately sexed and the effect that the sexing procedure may 

have on embryo viability (Taketo et al., 2005, Cenariu et al., 2008). 

The purpose of this paper was to evaluate the results obtained for bovine embryo sexing using 

the polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) and to compare 

them in order to decide which of the two methods is more accurate and yields better results while 

necessitating less effort.

Material and Methods

A total number of 152 bovine embryos have been used for sexing, and were divided into 2 

batches:

- batch 1, made up of 76 bovine embryos that were sexed using the polymerase chain reaction 

(PCR);

- batch 2, made up of 76 bovine embryos that were sexed using the fluorescent in situ hybridization 

(FISH).

The embryos have been non-surgically collected from a batch of 16 Simmental donor cows, 

following superovulation and artificial insemination. The embryos have been morphologically 

evaluated using a stereomicroscope and biopsy has been performed in order to collect a small number 

of blastomeres from the inner cell mass (fig.1). After biopsy, 

the embryos have immediately been transferred to recipient 

cows, whose estrous cycle had previously been synchronized 

with the donor cows.

The blastomeres obtained from batch 1 were submitted 

to DNA isolation and identification of certain nucleotide 

sequences found only on the Y chromosome, using specific 

primers. In short, the technique consisted of the following:

a. DNA extraction from the blastomeres using proteinase 

K;

b. Biosynthesis of the specific primers:

• One set of primers used for PCR were obtained using 

a bovine specific DNA sequence (1715 bovine satellite DNA) in order to show the presence of 

DNA in all the samples. Thus, the sequence of the two primers was: upstream 5’ – TGG AAG 

CAA AGA ACC CCG CT – 3’ downstream: 5’ – TCG TGA GAA ACC GCA CAC TG – 3’

• The second set of primers was obtained using the BRY4a repetitive sequence from the bovine 

genome that is highly specific for the Y chromosome and is present only in males. The sequence 

of the primers was upstream: 5’ – CTC AGC AAA GCA CAC CAG AC – 3’ and downstream: 5’ 

– GAA CTT TCA AGC AGC TGA GGC – 3’

c. Setting up of the PCR mixture that consisted of 10 ng DNA, 40 pmol of each primer and 45 µl of 

Platinum High Fidelity PCR Supermix (Invitrogen).

d. Amplification of the DNA sequences, using a thermocycler and the following amplification 

scheme: sample heating at 960C for 3 min., 33 cycles of denaturation at 950C for 1 min., primer 

annealing at 580C for 1 min. and primer extension at 720C for 1 min. and the final extension at 

720C for 5 min.;

e. Electrophoresis of the amplified samples in a 1.5% agarose gel stained with Gelstar Nucleic Acid 

gel stain;

Fig. 1 Aspiration of the blastomeres 
during embryo biopsy



21

f. Gel examination using a UV transilluminator:

• The presence of a single band corresponding to the bovine specific primers suggested the 

absence of Y-specific DNA sequences and thus made us classify the embryo as female;

• The presence of two bands one for the bovine specific primers and another for the Y-chromosome 

specific primers confirmed the presence of a Y-specific DNA sequence and thus made us classify 

the embryo as male.

The blastomeres obtained from batch 2 were treated with 1 µg/ml vinblastine sulphate for 6 

hours in order to induce the chromosomal metaphases, fixed on a slide using methanol and acetic 

acid and then kept in the freezer until use. The DNA probe was synthesized using the Y-chromosome 

specific BtY2 gene from which the following primers have been obtained: upstream 5’ - TGT TGT 

GAA GAA GGT GCC CA - 3’ and downstream 5’ - AGT TTG AGG GTG GTT GGT CG - 3’

The primers were used to amplify the male specific DNA sequence obtained after collecting blood 

from a bull and isolating the DNA from it. The amplification mixture consisted of 5 ng DNA, 1.5mM 

of each primer and 45 µl Platinum High Fidelity PCR Supermix (Invitrogen). The amplification 

conditions were: heating the mixture at 950C for 3 minutes followed by 33 cycles of denaturation 

at 950C for 30 seconds, primer annealing at 550C for 30 seconds and primer extension at 720C for 

30 seconds. The final extension consisted of keeping the samples at 720C for 7 minutes. After the 

amplification, the samples were run on a 1% agarose gel stained with Gelstar Nucleic Acid gel stain. 

Following electrophoresis, the gels were examined using a UV trans-illuminator, the bands were cut 

and the DNA was extracted from the gel in order to obtain the DNA probes used for fluorescent in 

situ hybridization. The DNA probes were biotinylated by nick translation.

The fluorescence in situ hybridization consisted of the following steps: rehydration of the 

blastomeres using decreasing concentrations of ethanol, target retrieval using heat and sodium 

citrate buffer, blastomere digestion using Triton X and proteinase K, fixation of the blastomeres 

using paraformaldehyde, application of the in situ frames, application of the hybridization buffer 

containing 1% DNA probe, hybridization reaction using a thermocycler (fig. 4) and the following 

hybridization scheme: 940C for 6 minute and 370C for 16 hours, washing the slides in 3 baths 

of PBS. The amplification of the fluorescent signal has been achieved using the tyramide signal 

amplification kit (Perkin-Elmer), which contains streptavidin-HRP, FITC conjugated tyramide and 

hydrogen peroxide. The slides were subsequently examined under a fluorescence microscope, in order 

to observe the green fluorescence in male embryos. The pregnancy rates obtained after the transfer 

of biopsied and sexed embryos were evaluated by rectal palpation, 60 days after the insemination, 

while the accuracy of embryo sexing was evaluated at birth, when the predicted sex was compared 

with the morphological sex of the newborn.

Results and Discussion

The embryo biopsy was successful for all of the embryos and the blastomeres were collected in 

good conditions.

After performing the experiences in the blastomeres belonging to batch 1, the following results 

have been obtained:

From the 76 embryos obtained, 70 (92.1%) had reached the morula stage, while 6 (7.9%) were in 

the early blastocyst stage. In what the quality of embryos was concerned, 73 embryos (96%) were 

transferable and were used for embryo biopsy while 3 (4%) could not be used for this purpose. 



22

After the amplification and gel electrophoresis of the 73 samples, the following results have been 

obtained: 

– 34 samples presented a single 216 bp DNA band when the bovine specific primers were used 

and no band when the Y-chromosome specific primers were used and thus they were considered 

to come from female embryos,

– 39 samples presented a 216 bp DNA band when bovine specific primers were used and a 301 

bp DNA band when Y-chromosome specific primers were used, thus being considered to come 

from male embryos.

When evaluating the pregnancy rate in recipient cows in which sexed embryos had been transferred, 

the following results have been obtained: 28 of 73 females have been diagnosed as pregnant, which 

represents a percentage of 38% (chart 1).

At birth, one of the calves obtained presented a 

different sex than the predicted one (female instead 

of male) which leads to an accuracy of 96.4% of the 

polymerase chain reaction sexing method.

After performing the experiences in the 

blastomeres belonging to batch 2, the following 

results have been obtained:

From the 76 embryos obtained, 72 (94.7%) had 

reached the morula stage, while 5 (5.3%) were in 

the early blastocyst stage. In what the quality of 

embryos was concerned, 74 embryos (97.4%) were 

transferable and were used for embryo biopsy while 

2 (2.6%) could not be used for this purpose.

The fluorescent in situ hybridization and the 

amplification of the fluorescent signal using the 

tyramide kit yielded the following results: 

– 41 samples presented the characteristic 

fluorescence and thus we considered the 

blastomeres to come from male embryos 

– 33 samples did not present any fluorescence 

and thus we considered the blastomeres to 

come from female embryos.

When evaluating the pregnancy rate in recipient 

cows in which sexed embryos had been transferred, 

the following results have been obtained: 30 of 74 

females have been diagnosed pregnant, which 

represents a percentage of 40.54% (chart 3).

At birth, 4 of the calves obtained presented 

a different sex from the predicted one (females 

instead of males) which leads to an accuracy of 

86.66% for the sexing of bovine embryos using the 

fluorescence in situ hybridization (chart 4).

73

28

45

0

10

20

30

40

50

60

70

80

recipient cows pregnant cows non-pregnant cows

Chart 1. Pregnancy rate in batch 1

Chart 2. Accuracy of the PCR method of bovine 
embryo sexing

28 27

1
0

5

10

15

20

25

30

total number of
calves obtained

correctly sexed
embryos

incorrectly sexed
embryos

74

30

44

0

10

20

30

40

50

60

70

80

recipient cows pregnant cows non-pregnant cows

Chart 3. Pregnancy rate obtained in batch 2



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Comparing the results obtained for the two 

methods of bovine embryo sexing, several obser-

vations can be made. First of all, the pregnancy 

rates obtained after transferring the biopsied 

embryos is almost similar for the two batches 

(around 40%), which is explainable as the biopsy 

technique did not differ between the two sexing 

methods. Improvements of the biopsy technique 

have to be made in the future in order to reduce 

the damage of the embryos and to be able to obtain 

higher pregnancy rates. In what the accuracy of the 

two methods is concerned, the PCR sexing yielded better results than the FISH method, being at the 

same time easier to perform and requesting lower expenses for materials and equipments.

Conclusions

1. The pregnancy rates obtained after transferring the biopsied embryos were of 38% in batch 1 
and 40.54% in batch 2, being almost similar as the biopsy technique did not differ between the two 
sexing methods.

2. The accuracy of the PCR method of bovine embryo sexing was of 96.4%, one of the embryos 
having a different sex than the predicted one.

3. The accuracy of the FISH method of bovine embryo sexing was of 86.66%, four of the embryos 
having a different sex than the predicted one.

4. The PCR sexing of bovine embryos yielded better results than the FISH method, being at the 
same time easier to perform and requesting lower expenses for materials and equipments.

5. Improvements of the biopsy technique have to be made in the future in order to reduce the 
damage of the embryos and to be able to obtain higher pregnancy rates.

6. We recommend the commercial use of PCR sexing kits for bovine embryo sexing, the FISH 
method being appropriate only for research purposes.

References

1. Bredbacka P. – Progress on methods of gene detection in preimplantation embryos. Theriogenology 55 
pp 23-34, 2001.

2. Cenariu M., I. Groza, R. Al. Pop, Brînduşa Stegeran, Emoke Pall, Laura Cătană, A. Bartoş - Bovine embryo 
sexing using the fluorescence in situ hybridization (FISH), Bulletin of the University of Agricultural 
Sciences and Veterinary Medicine Cluj-Napoca, Veterinary Medicine, 65 (2)/2008, ISSN 1843-5270, 
pp 109-113, 2008.

3. Cenariu M., I. Groza, Simona Ciupe, Brânduşa Stegeran, Emoke Pall, Cătană Laura, A. Bartoş - Bovine 
embryo sexing using the polymerase chain reaction (PCR), Scientific Papers USAMV Iaşi, Veterinary 
Medicine, vol. 51/2008, ISSN 1454-7406, pp 252-256, 2008

4. Groza I., L. Bogdan, I. Morar, Simona Ciupe, R. Pop, M. Cenariu, C. Peştean - Cercetari privind variantele 
de inovulare a embrionilor la bovine, Clujul Medical Veterinar, nr.8, pp 8-12, 2005.

5. Groza I. şi col. – Ginecologie, Andrologie şi Obstetrică Veterinară, Ed. Academiei Române, Bucureşti, 2006
6. Lee J.H., J. H. Park S.H. Lee, C.S. Park, D. I. Jin - Sexing using single blastomere derived from IVF bovine 

embryos by fluorescence in situ hybridization (FISH), Theriogenology 62, pp 1452-1458, 2004
7. Jin L., R.V. Lloyd - In situ hybridization: methods and applications. J.Clin. Lab. Anal. 11(1), pp 2-9, 1997
8. Shea B.E. - Determining the sex of bovine embryos using polymerase chain reaction results: a six-year 

retrospective study. Theriogenology 51, pp785-797, 1999.
9. Taketo, T., Lee, C.H., Zhang, J., Li, Y., Lee, C.Y., Lau, Y.F. - Expression of SRY proteins in both normal 

and sex-reversed XY fetal mouse gonads. Dev. Dyn. 233, pp 612–622, 2005.

30
26

4

0

5

10

15

20

25

30

total number of
calves obtained

correctly sexed
embryos

incorrectly sexed
embryos

Chart 4. Accuracy of the FISH bovine embryo sexing method


