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Research Article

Molecular Detection of Polymorphism of Heat Shock Protein 70 (Hsp70) in the Semen of Iraqi Holstein Bulls

Hassan Nima Habib, Amad Falah Hassan and Bassam Yasein Khudaier
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Background and Objective: Hsp70 is a molecular chaperone and cell preservation against heat shock, considered as the ideal biological marker for measuring heat stress in animals, its polymorphism may affect stress tolerance bulls’ fertility. The study aimed to identify polymorphism of hsp70 gene in Holstein bulls born in Iraq. Materials and Methods: The study was carried out during 1st November, 2015 to 31th January, 2016, 29 Holstein bulls born in Iraq, 2.5-3.5 years old, back to Artificial Insemination Center of Abou-Ghareeb Baghdad, Iraq, were used. Semen was collected by using the artificial vagina. DNA extracted then PCR amplified, sequencing, BLAST analysis and multiple sequence alignment were carried out. Results: The results showed three haplotypes, haplotype G1, haplotype G2 and haplotype G3 as a compare with the same gene in GenBank, they all showed silent mutations in different positions except haplotype G2 showed a missense mutation in position 1451 as well as silent mutations. Conclusion: It is concluded that the Holstein bulls born in Iraq have new polymorphism of Hsp70 gene as a compare with same gene in GenBank, because of mutations.

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  How to cite this article:

Hassan Nima Habib, Amad Falah Hassan and Bassam Yasein Khudaier, 2017. Molecular Detection of Polymorphism of Heat Shock Protein 70 (Hsp70) in the Semen of Iraqi Holstein Bulls. Asian Journal of Animal Sciences, 11: 132-139.

DOI: 10.3923/ajas.2017.132.139

Received: March 15, 2017; Accepted: April 06, 2017; Published: April 15, 2017

Copyright: © 2017. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.


Heat shock protein 70 (Hsp70) is one of HSPs family and its size is 68-73 kDa1. It is encoded by a single exon of the Hsp70 gene, the open reading frame to this gene is 1926 bp approximately. The protein comprises 641 amino acids2. The work of Hsp70 is molecular chaperones and cell preservation against heat shock, able of denaturing proteins3, it's one of the most important roles is prevent aggregation of the partially folded proteins remodel folding pathways and regulate activity of proteins4. Expressed in response to heat stress and environmental stress5, hsp70 gene was considered as the ideal biological marker for measuring heat stress in animals6. Polymorphism of hsp70 gene explains the differences between individuals in the tolerance of stress conditions such as heat stress, polymorphism produced by genetic mutations which can be a useful to resist heat stress7. Nucleotide polymorphisms in the hsp70 gene promoter region may affect stress tolerance and haplotypes of Hsp70 were associate with bulls’ fertility and heat tolerance8,9, the difference in reproductive performance was also associated with polymorphism of hsp70 gene10. The Hsp70 is available in spermatozoa of mature bulls11. Although the origin of the Holstein cattle is the cold regions (Europe), can be bred and preserve in tropical and sup-tropical environment like Iraq12, Hsp70 playing a pivotal function in environmental and heat stress adaptation13,14, because it is playing important roles in essential cellular processes and also related to stress15. The main objective of this study was to identify the manipulation of genotype (molecular changing) to capable with harsh environment (high temperature). Therefore, polymorphisms of the hsp70 gene of the Holstein bulls born in Iraq were identified. Since there have been no studies and the lack of information dealing with this subject, this study aimed to identify polymorphism of Hsp70 gene in Holstein bulls which are born in Iraq.


Animals and semen collection: The study was carried out during 1st November, 2015 to 31th January, 2016. Twenty nine Holstein bulls born in Iraq of known fertility, 2.5-3.5 years old, back to Artificial Insemination Center of Abou-Ghareeb Baghdad, Iraq were used in the present study. Semen was collected from all bulls by using the artificial vagina method.

DNA extraction: The DNA was extracted from semen samples using Chelex-100® (Sigma Aldrich, USA) as already described by Walsh et al.16 and modified by Manuja et al.17. Concentration and purity of DNA was estimated by using Nano-drop system (Nano Drop thermo scientific 200, USA). To determine the best purity, with an estimate 260/280 ratio, which should be 1.8, that indicate the purity of the DNA18.

PCR amplification: Amplifications were performed in 25 μL reactions (Table 1). According to First BASE Laboratories/Malaysia, primer was used to amplify conserved region within the HSP70 promotor, Hsp70-F: ATGGCGAAAAACATGGCTATCGGC, hsp70-R: CTAATCCACCTCCTCAATGGTGGGGCC, First BASE Laboratories/Malaysia, the PCR ( A-2040-1 My Genie/Gradient Thermal Bioneer Korea) amplification cycling protocols of PCR amplification are summarized in Table 2. The PCR product was detected on 1.5% ethidium bromide stained agarose gel. PCR product size was 1926 bp (Fig. 1).

Sequencing: The PCR products purification, then sequenced in First BASE Laboratories/Malaysia. Nucleotide sequence alignments and comparisons were done using the Geneious version 10.1.3 software. BLAST analysis were carried out on website, Multiple Sequence Alignment were carried out on website and compared with same gene in Gene Bank accession number AY662497.1.


Results of sequencing and the multiple sequence alignment were compared with heat shock protein Hsp70 gene of the Holstein bulls in the gene bank. Three new haplotypes were detected and classified as three groups.

First group, the haplotype G1, is the closest haplotype to heat shock protein hsp70 gene in the Holstein bulls in the gene bank.

Table 1:Components of PCR reactions
Image for - Molecular Detection of Polymorphism of Heat Shock Protein 70 (Hsp70) in the Semen of Iraqi Holstein Bulls

Table 2:Cycling protocol of PCR amplification
Image for - Molecular Detection of Polymorphism of Heat Shock Protein 70 (Hsp70) in the Semen of Iraqi Holstein Bulls

Image for - Molecular Detection of Polymorphism of Heat Shock Protein 70 (Hsp70) in the Semen of Iraqi Holstein Bulls
Fig. 1:Gel electrophoresis of PCR products of Hsp70 gene
Sample 1 and 7: DNA marker, Sample 2: -ve is no-template control (water to replace DNA template), Sample 3-6: DNA template (Hsp70 gene), A total 75 ng of DNA sample was used in one 25 μL PCR reaction. only 3 μL of PCR product was run on 1% TAE agarose of PCR product was run on 1% TAE agarose gel at 100 V, 60 min

This haplotype was exposed to genetic mutations in nucleotides at positions, 6 (C<G), 174 (C<G), 282 (A<G) and 1339 (T<C). All mutations are silent that did not encode a new amino acid. Fifteen Bulls were found in this group.

Second group, the haplotype G2, in comparison with Hsp70 gene in the Holstein bulls in the gene bank and first group. Nucleotides were substituted in eight different positions, 114 (G<A), 174 (C<G). 282 (A<G) ,1339 (T<C), 1451 (C<A), 1590 (A<G ), 1695 (C<T) and 1719 (G<T). These genetic mutations are all silent except the mutation at the position 1451 (missense mutation). It encoded for new amino acid (Aspartic acid instead of alanine). The change nucleotide cytosine (C) instead of adenine (A) led to a change code GCC to GAC which encode a new amino acid19. Six bulls were found in this group.

Third group, the haplotypeG3, in comparison with Hsp70 gene in the Holstein bulls in the gene bank and other groups, in this group nucleotides were substituted for each position 114 (G<A), 174 (C<G), 282 (A<G), 1339 (T<C), 1590 (A<G), 1695 (C<T) and 1719 (G<T). All mutations are silent. Eight bulls were found in this group.

The results of the Multiple Sequence Alignment (MSA) of hsp70 gene in the bulls (Fig. 2) showed that the convergence ratio of G1, G2 and G3 haplotypes were 99.73, 99.51 and 99.56%, respectively.

These results showed silent mutations in different positions of hsp70 gene, although it does not encode a new amino acid20 but it may influence the relevant protein through change in transcription and may impact the precision or efficiency of splicing of mRNA or control of transcripts21,22. On the other hand the missense mutation that occurred in haplotype G2 may change protein properties due to differences between amino acids, aspartic and alanine23.

These mutations may effect on protein folding and stability24, protein function25 and protein-protein intractions26.

Image for - Molecular Detection of Polymorphism of Heat Shock Protein 70 (Hsp70) in the Semen of Iraqi Holstein Bulls
Image for - Molecular Detection of Polymorphism of Heat Shock Protein 70 (Hsp70) in the Semen of Iraqi Holstein Bulls
Image for - Molecular Detection of Polymorphism of Heat Shock Protein 70 (Hsp70) in the Semen of Iraqi Holstein Bulls
Fig. 2:Identified Hsp70 gene in Iraqi Holstein bulls and the same gene in GenBank
  CLUSTAL Omega (1.2.4) multiple sequence alignment

The result agree with Li et al.27, Sodhi et al.28, Kerekoppa et al.29 and Bhat et al.3 on the possibility of polymorphism of hsp70 gene in cattle.

While the results indicate that the Iraqi Holstein bulls have new haplotypes of hsp70 in comparison with that recorded in GenBank. The haplotypes of this gene identified from Holstein cattle in Japan and Poland were identical accession number AY662497.130,31. Similar results were found in Italian6 and Chinese Holstein32. However, polymorphism of crossing Indian cattle with Holstein disagreed with this result33.

There is no study in line with the results of this study on the location of mutations and perhaps the reason is that this study is the first of its kind on the hsp70 gene polymorphism in the Holstein bulls in Iraq.

These results might indicate that the Iraqi Holstein bulls have adapted significantly to resist heat stress conditions through changes in the polymorphism of the Hsp70 gene, that's because the mutations in Hsp70 gene could be used as marker for heat stress tolerances27 and assisted selection for resistant to heat stress in the breeding34 but this needs to be further studied to determine the association between Hsp70 gene polymorphism in semen and its characteristics in Holstein bulls born in Iraq.


This study showed three new haplotypes of the Hsp70 gene in the Holstein bulls born in Iraq as a result of mutations in different positions, where Holstein can modify their genotypes of hsp70 gene to adapting surrounding environmental conditions, even it was originated in different environments. These changes did not affect the animal fitness.


This study discovers that the Hsp70 gene can manipulate by mutations (silent or missense) according to the environment conditions. This study will help the researcher to uncover the polymorphisms of hsp70 gene in Holstein bulls born in Iraq and helps to study the relationship between hsp70 gene polymorphism and semen quality as a guide to the selection of bulls.


The authors would like to thank Prof. Dr: Asaad Y. Ayied, Department of Animal Production, College of Agriculture, University of Basrah, Iraq. For his scientific assistance in completing this paper.


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