Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 21-30 Abdulmuttaleb Hazim khaleel, Salih Hassan Al-Azzawi ISSN: 2073-9524 eISSN: 2310-8746 21 Polymorphisms Detection in Heat Shock Brotein70 Gene (HSP70) and its Association with Semen Quality in Local Iraqi Goats Abdulmuttaleb Hazim khaleel 1 , Salih Hassan Al-Azzawi 2 1,2 Department of Animal Production - College of Agriculture University of Diyala- Iraq 1 Corresponding author: abdmtaleb85@gmail.com Article history: Received: 27 December 2021 Accepted 13 March 2022 Published: 30 June 2022 Abstract Heat shock protein 70 (HSP70) is a vital gene affecting semen quality traits. The study aimed to estimate the breeding values additive dominance variance of Iraqi black goat semen quality and investigate the effect of allele substitution in heat shock protein 70 gene (HSP70) carpine on some semen quality traits (volume, mass motility, a live sperm, and sperm concentration). DNA isolated from 15 Iraqi black goats was subjected to PCR amplification of the caprine HSP70 gene. Used Single-nucleotide polymorphism (SNP) to detect the variant DNA fragments that were sequenced. Synonymous Genotypes were detected on mutation locus (1528C/T). Results showed three genotypes (CC, CT, TT) frequencies of 0.53, 0.40, and 0.07, respectively, and gene frequency of 0.73 and 0.27 C and T, respectively. The population was in Hardy Weinberg equilibrium. The results showed a high level of concentration sperm for CC and CT genotype compared with TT genotype. The CC genotype was associated with negative B.V for volume ejaculate, mass motility, and live sperm percentages and was positively associated with sperm concentration. CT and TT genotypes showed positive B.V for volume ejaculate, mass motility, and live sperm but negative for sperm concentration. CC and TT genotypes showed negative dominance deviation for all traits studied, while heterozygous genotype CT showed positive dominance deviation for all semen quality traits. The highest genetic variation for live sperm (0.816) but most of this variation is from dominance variance (0.653), followed by Genetic variance for mass motility (0.481), most of it from Additive variance (0.39). Keywords: Breeding value, Dominance variance, alleles substitute, semen, HSP70 gene, local Iraqi goats. https://dx.doi.org/10.52951/dasj.22140103 This article is open access under the CC BY 4.0 license (http://creativecommons.org/licenses/by/4.0/). Introduction Domestic goats are essential genetic resources that adapt to local conditions and can be used to provide meat and milk to consumers. Local goat breeds may be used as sires or even dams for crossing with foreign breeds to produce hilly tolerant crosses to harsh environmental conditions and productivity (Al-Qudsi and Al- Rawi,2021; Khalil and Jassim,2018). The new Molecular genetics techniques enable the researchers to use genes of high economic importance found in certain breeds to be transferred to others) Khazraji et al., 2020). Heat shock proteins (HSPs) are a group of proteins produced by the cells in response to stress conditions and are considered one of the most critical responses to stress where HSPs genes are activated (Habib and Saleh, 2019). The HSP70 gene consists of 1926 base pairs encoding the 641 amino acids found in the HSP70 protein of sheep and goats (Gade et al.,2010; Pawar et al.,2013). HSP70 gene in goats is located on chromosome 23 (Raza et al.,2021) HSP70 mitigates the effects of heat stress on cells by mitigating or preventing damage to proteins (Miova et al., 2015). HSP70 prevents the harmful effects of heat stress by inhibiting apoptosis, and this is the internal mechanism by which animals adapt to high mailto:abdmtaleb85@gmail.com http://creativecommons.org/licenses/by/4.0/ https://orcid.org/0000-0001-9472-9203 https://orcid.org/0000-0002-9105-6477 https://orcid.org/0000-0001-9472-9203 https://orcid.org/0000-0002-9105-6477 Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 21-30 22 environmental temperatures (Mishra and Palai, 2014). In mice, high expression of HSP70 protein limits testicular degeneration, confirming its protective role against high temperature (Kon and Endoh, 2001). The heat shock proteins HSP70 affect the characteristics of an organism's semen, as they bind to newly formed proteins and assist in the folding and synthesizing of proteins during spermatogenesis (Dun et al., 2012). It helps to deal with the negative effects of various stress conditions, including high temperatures (Gullo and Teoh, 2004), that a polymorphism in the hsp70 gene was associated with the sperm quality of pigs in the hot season (Huang et al., 2002). and the characteristics of bull sperm (Shrum et al., 2010). The polymorphism in the HSP70 gene in bulls semen affects its quality. It is possible its use in artificial insemination programs as a marker that helps select good bulls for insemination (Gafer et al., 2015). Genetic improvement using selection by genetic markers and genome information is primarily based on the additive effect, which plays an essential role in influencing the physiology and evolution of farm animals (Cloete et al., 2004). Understanding the genetic characteristics of a herd requires an analysis of components of Additive variance (VA), which represents breeding value, dominance variance (VD), and supra- dominant variance (VI) (Falconer and Mackay, 1996). This study aims to estimate the breeding values, calculate the total genetic variance and determine the additive and dominant variability as well as the effect of substitution of alleles and the average effect allele for locus mutation (1528C/T) of the HSP70 gene in Iraqi Bucks and their association with seminal traits (Volume and mass motility, sperm concentration and a live sperm). Material and Methods Ethical approval The Scientific Ethical Committee of the animal production department College of Agriculture University of Diyala, Iraq, approved this study (22/3/2021). Experimental animals: This study was conducted in Iraq at the University of Diyala, on 15 Iraqi Bucks average age (1-2) year the average weight of 29 kilograms from 15/6/2021to 15/9/2021. It was placed in a semi-shaded barn, and it was fed a quantity of 500 grams per each Bucks, provide concentrated containing in the rate of 33% of the bran and 65% of barley, and the mixture was supplemented by adding the rate of 1% salt and 1% a mixture of vitamins and minerals, hay as a rough feed available most of the time. Semen collection Semen was collected using an artificial vagina in the morning at a rate of six ejaculates per buck tucked biweekly for three months. The semen was contained in a graduated glass tube where the semen volume was measured immediately after collection. Then the glass tube was placed in a water bath (temperature of 37 °C) found in the field laboratory. Mass motility was estimated by placing a drop of semen on a glass slide at 37°C under a microscope at 10× magnification. The wave motion was estimated based on the strength and intensity of moving waves (Walton, 1933). The percentage of live sperm was calculated according to the method of (Swanson and Bearden, 1951). Deads sperm examined under 400× microscopic magnification concentration was calculated according to the method (Salisbury et al., 1943). Laboratory side: DNA.extraction: Blood samples were taken from the jugular vein, the blood was placed in test tubes containing anticoagulant EDTA) to extract DNA according to the (ABIOpure) protocol of the Advance Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 21-30 23 Scientific Company (ASCO) located in Iraq / Baghdad city (Al- Harithiya) and gene isolation Heat shock (HSP70) for sequence analysis for the period from 9/18/2021to 10/21/2021. Primers design and the polymerase chain reaction The studied part was identified by Primers shown in table (1), which were designed by the Advance Scientific Company (ASCO) using the program Primer3 belonging to the National Center for Biotechnology Information )NCBI( were supplied by a Korean company (Macrogen). The molecular detection of the HSP70 gene was carried out using the polymerase chain reaction technique using the reaction mixture consisting of the components detailed in Table (3) according to the program shown in table (2) Table 1. Primers sequence used in the study Primers sequence Primer Name Start Stop Amplicon size (pb) Gene name Annealing temp Cº Forward (Sense) 5-GACCTCAACAAGAGCATCAA -3 F 1432 1452 903 pb HSP70 65 Reverse (Anti Sense) 5-GATCCCAACAGTCTCCATAAC-3 R 2314 2335 Table 2. The PCR amplification program Seq Steps temperature C 0 Time Number cycle 1 Initial Denaturation 95 5 minutes 1 Cycle 2 Denaturation 95 30 second 30 Cycle 3 Annealing 65 30 second 4 Extension 72 30 second 5 Final Extension 72 7 minutes 1 Cycle Hold 10 Table 3. Components of the polymerase chain reaction mixture Components used for PCR reaction volume Master Mix 12.5µl Forward primer 1 µl Reverse primer 1 µl Nuclease Free Water 7 µl MgCl2 0.5µl DNA 3 µl Total volume 25 µl Polymerase chain reaction and Electrophoresis: Alagros Gel electrophoresis was used To check for PCR products. The electrical power for Electrophoresis was turned on at 100 volt / 50 mAmp for 60min. The electrophoresis product was a figure (1). The nucleotide sequence was determined by sending the polymerase chain reaction product to the Korean company (Macrogen) through the Advance Scientific Company. Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 21-30 24 Fig. 1. The Electrophoresis of the product (PCR) of portion (HSP70) gene size 903 bp, which was studied for 15 samples of Iraqi bucks Statistical analysis: The genotypic frequencies and allelic frequencies were calculated using the SAS (2012) Version 9.1 program. The genotype distribution in this population was tested for Hardy-Weinberg using the appropriate Chi-square (χ2) test. The equations for each calculated value were applied as follows (Falconer and Mackay ، 1996): 1- Average effect of allele C = q [a +d (q – p) = αC 2- Average effect of allele T = - p[a +d (q – p) = αT 3- Effect of substitution of alleles = αC - αT 4- Breeding values : CC = 2 αC ,CT = αC + αT ,TT = 2 αT 5- Dominant deviations : CC = -2q 2 d , CT = 2pqd , TT = 2p 2 d 6- Different variances : VA = 2pq α 2 , VD = 4 p 2 q 2 d 2 , VG = VA + VD Results and Discussion Percentages of genotypes and allelic in heterogeneity locus of sequences nitrogenous base (C1528T) in HSP70 gene for 15 samples of Iraqi bucks The analysis using Sequences technology revealed heterogeneity in the gene (HSP70) at the locus (1528 C/T), and the genetic makeup of the experimental animals was determined as shown in table (4). It contained three genotypes CC (Wild), CT (heterozygous), and TT (recessive), as shown in Figure (2). Results showed that the nitrogen base was changed from C to T (1528 C/T). The percentage heterogeneity locus genotypes were 53, 40, and 7% for the genotypes CC, CT, and TT, respectively, with an overall genes frequency of 0.73 for C and 0.27 for the T allele. The values of the chi-square test (x 2 ) showed that all genotypic frequencies in the population were in Hardy Weinberg equilibrium (p>0.05), indicating the selection pressure on this site in the population was not too powerful. Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 21-30 25 Fig. 2. The location of the variation (1528) in the third segment of the heat shock gene (HSP70) for a sample of local male Iraqi goats, where the letter (Y) symbolizes the fact that the mutation (transition mutation) where the nitrogen base (C) of the type pyrimidine was replaced by a nitrogen base (T) It is also a type of pyrimidine. Table 4. Number and percentage for the genotype of locus sequences nitrogenous base (1528 C/ T) in gene HSP70 for 15 samples of Iraqi bucks Percentage% Number Genotype 53.00 8 CC 40.00 6 CT 7.00 1 TT 100 15 SUM 0.038 Chi-square ( χ 2 ) value Allelic Frequency 0.73 C 0.27 T NS P>0.05 There was no difference between the average genotypes of volume ejaculate, mass motility, and live sperm. As for the concentration of sperm, there was a significant difference between the genotypes (Table5). Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 21-30 26 Table 5. Relationship of genotype (1528 C/T ( with some fresh semen traits (mean and standard error) of volume ejaculate, Mass motility, live sperm, and sperm concentration Trait Genotype CC CT TT Volume ejuculate 0.9075+ 0.032 a 0.945+ 0.035 a 0.97+ 0.152 a Mass motility 76.56 + 1.130 a 77.97+ 1.277 a 77.83+ 2.891 a live sperm 82.75+ 0.700 a 84.5+ 1.051 a 82.15+ 2.011 a Sperm concentration 1.72 + 0.023 ab 1.77+ 0.031 a 1.63+ 0.037 b Different letters within the same row indicate a significant difference between the means at the probability level (0.05). The results of the mean allele substitution in Table (6) showed that the mean substitution of the allele (T) instead of the allele (C) in this study is (0.034, 0.992, 0.643) for volume ejaculate, mass motility of sperms, and percentage of alive sperms respectively, and mean allele substitution (C) At the allele locus (T) is (- 0.034,- 0.992,-0.643) which means that selection towards the allele (T) is essential to improve these traits. But regarding sperm concentration, It was the mean substitution of the allele (C) instead of the allele (T) in this study is (0.0004). The mean allele substitution (T) at the allele locus (C) is (- 0.001), which means that selection towards the allele (C) is essential to improve this trait. Table 6. Mean genetic effect and mean effect of genetic substitution of alleles for semen traits (Volume ejaculate, Mass motility, live sperm, and Sperm concentration) for the (1528 C/ T) locus in the HSP70 gene Trait Alleles Average gene effect The average effect of gene substitution Volume ejuculate C -0.009 -0.034 T 0.025 0.034 Mass motility C -0.268 -0.992 T 0.724 0.992 live sperm C -0.174 -0.643 T 0.469 0.643 Sperm Concentration C 0.0004 0.001 T -0.001 -0.001 Effect of the genetic polymorphism of (1528C/T) of genetic variance component shown in Table (7) Bucks with (TT) and (CT) genotypes had higher mean ejaculate volume and Mass motility where the average ejaculate volume was (0.97 and 0.945 ml,) respectively and mass motility (77.97, 77.83%) respectively compared to the (CC) genotype where the average ejaculate volume was (0.9075 ml) and the Mass motility (76.56%). The genotypes (TT) and (CT) with the highest breeding value compared to genotype (CC). Therefore, it is possible to choose the genotype (TT, CT) to improve the characteristics of the semen (ejaculate volume and mass motility) because the breeding values are the value that can be Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 21-30 27 transferred to future generations (children) (Warmington and Kirton, 1990). That individuals with the (CT) genotype have the highest mean percentage of live sperms, which was (84.5%) compared to the (CC) and (TT) genotypes, where the average rate of live sperms was (82.75 and 82.15%) respectively and genotype (TT, CT) which is the highest breeding value where it was (0.939 and 0.296) respectively, and that TT genotype is the least dominance deviation compared to genotype (CC, CT) and where the genotype (CT) It had the highest average percentage of live sperms and that its breeding values (0.296) are positive, which can be transmitted to future generations (Warmington and Kirton,1990). where the average effect of the allele and the mean substitution of the allele (T) was positive for these traits compared to the allele (C), which was negative. Therefore, it is possible to select the genotype (CT) to improve the trait (live sperm). That the genotype (CT) recorded the highest average sperm concentration, which was (1.77×10 9 /ml) with a significant difference from the two genotypes (TT, CC), where the average sperm concentration was (1.72 and 1.63×10 9 /ml), as for the values of breeding, they were low for all structures, where the highest value was (0.001) for the genotype (CC). For the two genotypes (TT, CT), the Breeding value was (-0.001, -0.002), respectively, and the lowest sovereign deviation was recorded for the genotype (CC). Followed by the two genotypes (TT) and (CT), it was (-0.014, - 0.101, 0.037) respectively. The genotype (CT) recorded the highest average sperm concentration. Still, its low breeding value may indicate a low probability of transmission of this trait as it was the average effect of the allele and the mean substitution of the allele (T) is negative for these traits compared to the allele (C), which was positive. Nikbin et al. (2014) reported only one study; two mutations were recorded at the site (74A>C) (ss836187517), and genotypes were AA, AC, CC site and (191C>G) (ss836187518) and genotypes were CC, CG, GG. The C variant allele at the (74A>C) mutation site and the G variant at the (191C>G) mutation site were more frequent. In contrast, the T allele was less frequent in the current study. In the study of Nikbin et al. (2014), it was found at the mutation site (191 C>G) that the pure genotypes (CC, GG) outperformed the heterozygous (CG) in (sperm concentration, mass motility, and live sperm) while the ejection size outperformed the heterozygous (CG) on the pure genotypes. As for the mutation site (74 A>C), the AC genotypes outperformed the pure genotypes (CC, AA) in sperm concentration. As for the (mass movement and live sperm), the heterozygous AC genotypes were the least significant. It is close to the result of the current study, where the genotype CT was significantly better in sperm concentration. In a study by Nikbin et al. (2014), it was found that the C allele of the mutation (191C>G) had a positive effect on sperm concentration, as well as the site of the mutation (74 A>C) .The C allele was the best in increasing the sperm concentration in the semen, and this result is close to the result of the current study, where the C allele was the best in increasing the sperm concentration .This study could be an excellent way to select animals, but more studies are needed to prove this assumption. Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 21-30 28 Table 7. Breeding value, dominance deviation, and genetic variations genotypes for the (1528 C/T) locus in the HSP70 gene for semen traits (volume ejaculate, mass motility, live sperm, and spermconcentration) Volume ejaculate Genotype No Mean BV DD VA VD VG CC 8 0.9075 -0.018 -0.001 0.0005 0.00001 0.0005 CT 6 0.945 0.016 0.002 TT 1 0.97 0.050 -0.007 Mass motility Genotype No Mean BV DD VA VD VG CC 8 76.56 -0.535 -0.113 0.39 0.0933 0.481 CT 6 77.97 0.456 0.306 TT 1 77.83 1.448 -0.826 live sperm Genotype No Mean BV DD VA VD VG CC 8 82.75 -0.347 -0.299 0.163 0.653 0.816 CT 6 84.5 0.296 0.808 TT 1 82.15 0.939 -2.185 Sperm concentration Genotype No Mean BV DD VA VD VG CC 8 1.72 0.001 -0.014 0.000001 0.0014 0.001 CT 6 1.77 -0.001 0.037 TT 1 1.63 -0.002 -0.101 BV: Breeding value, DD: Dominance deviation, VA: Additive variance, VD: Dominance variance, VG: Genetic variance. Conclusion Heat shock protein 70 (HSP70) is a vital gene affecting semen quality traits.The estimate the breeding values additive dominance variance of Iraqi black goat semen quality and investigate the effect of allele substitution in heat shock protein 70 gene (HSP70) carpine on some semen quality traits. Results showed three genotypes (CC, CT, TT). The results showed a high level of concentration sperm for CC and CT genotype compared with TT genotype. The CC genotype was associated with negative B.V for volume ejaculate, mass motility, and live sperm percentages and was positively associated with sperm concentration. CT and TT genotypes showed positive B.V for volume ejaculate, mass motility, and live sperm but negative for sperm concentration. CC and TT genotypes showed negative dominance deviation for all traits studied, while heterozygous genotype CT showed positive dominance deviation for all semen quality traits. Acknowledgments The authors thank Dr. Bashar Adham (head of the department) and Dr. Zaid Muhammad Mahdi at Diyala University, College of Agriculture, Department of Animal Production, for their assistance. Conflict of interests The authors declare that they have no competing interests. Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 21-30 29 References Al-Qudsi, N.H., Al-Rawi, A.A.(2021) Strategy for the development of Iraqi animal genetic resources based on climate changes. Ministry of Higher Education and Scientific Research, Republic of Iraq. In Arabic. Cloete, S. W. P., Gilmour, A. R., Olivier, J. J., and Van Wyk, J. B. (2004). Genetic and phenotypic trends and parameters in reproduction, greasy fleece weight and liveweight in Merino lines divergently selected for multiple rearing ability. Australian Journal of Experimental Agriculture, 44(8), 745- 754. Dun, M. D., Aitken, R. J., and Nixon, B. (2012). The role of molecular chaperones in spermatogenesis and the post-testicular maturation of mammalian spermatozoa. Human reproduction update, 18(4), 420-435. Falconer, D. S., and Mackay, T. F. C. (1996) . Introduction to quantitative genetics 4th edition. Harlow, UK: Longmans. Gade, N., Mahapatra, R. K., Sonawane, A., Singh, V. K., Doreswamy, R., and Saini, M. .(2010). Molecular characterization of heat shock protein 70-1 gene of goat (Capra hircus). Molecular biology international. Gafer, J. A., El-Rahman, G. H. A., and Rawash, Z. M. (2015). Association of Hsp70 Gene Polymorphism and Bull Semen Quality in winter and Summer Seasons. Alexandria Journal for Veterinary Sciences, 46(1). Gullo, C. A., and Teoh, G. (2004). Heat shock proteins: to present or not, that is the question. Immunology letters, 94(1-2), 1-10. Habib, H. N., and Saleh, W. M. M. (2019). The Role of Heat Shock Proteins 70 (HSP70) in Farm Animals Adaptation, A Review Paper. In The 2nd International Scientific Conference, Qurna Education College. Huang, S. Y., Chen, M. Y., Lin, E. C., Tsou, H. L., Kuo, Y. H., Ju, C. C., and Lee, W. C. (2002). Effects of single nucleotide polymorphisms in the 5′- flanking region of heat shock protein 70.2 gene on semen quality in boars. Animal Reproduction Science, 70(1-2), 99-109. Khalil Z. S., and Jassim, S. H., (2018). Estimate of genetic parameters and some non-genetic factors to produce milk and its components in the local and shami goats centeral Iraq. Diyala Agricultural Sciences Journal, 10(2), 26-35. Khazraji, W. J. A., AL-Khuzai, H. M., and AL-Shaikh, M. A. (2020). Analysis of Genetic Variance for Milk Production And it Components in Local Goat For Prolactin Gene. Diyala Journal of Agricultural Sciences, 12. (A special issue of the proceedings of the Fourth Scientific Conference on Agricultural Research). Kon, Y., and Endoh, D. (2001). Heat‐shock resistance in experimental cryptorchid testis of mice. Molecular reproduction and development, 58(2), 216-222. Miova, B., Dinevska‐Kjovkarovska, S., Esplugues, J. V., and Apostolova, N. (2015). Heat stress induces extended plateau of Hsp70 accumulation–a possible Cytoprotection mechanism in hepatic cells. Journal of cellular biochemistry, 116(10), 2365-2374. Mishra, S. R., and Palai, T. K. (2014). Importance of heat shock protein 70 in livestock-at cellular level. J Mol Pathophysiol Apr-Jun, 3(2). Diyala Agricultural Sciences Journal Vol (14) No 1, 2022: 21-30 30 Nikbin, S., Panandam, J. M., Yaakub, H., Murugaiyah, M., and Sazili, A. Q. (2014). Novel SNPs in heat shock protein 70 gene and their association with sperm quality traits of Boer goats and Boer crosses. Animal reproduction science, 146(3-4), 176-181. Pawar, H. N., Agrawal, R. K., and Brah, G. S. (2013). Expression, purification and characterization of recombinant Heat Shock Protein 70 (HSP70) from sheep and goat species. International Journal of Current Microbiology and Applied Sciences, 2(11), 440-452. Raza, S. H. A., Hassanin, A. A., Dhshan, A. I., Abdelnour, S. A., Khan, R., Mei, C., and Zan, L. (2021). In silico genomic and proteomic analyses of three heat shock proteins (HSP70, HSP90-α, and HSP90-β) in even-toed ungulates. Electronic Journal of Biotechnology, 53, 61-70. Salisbury, G. W., Beck, G. H., Elliott, I., and Willett, E. L. (1943). Rapid methods for estimating the number of spermatozoa in bull semen. Journal of Dairy Science, 26(1), 69-78. SAS. (2012). Statistical Analysis System, User's Guide. Statistical. Version 9.1th ed. SAS. Inst. Inc. Cary. N.C. USA. Shrum, K., Lester, T., Rorie, R., Reiter, S., Looper, M., and Rosenkrans Jr, C. (2010). Effects of heat shock protein 70 haplotype and tall fescue variety on bull sperm characteristics. Arkansas Agricultural Research Station Research Series, 584, 39-44. Swanson, E. W., and Bearden, H. J. (1951). An eosin-nigrosin stain for differentiating live and dead bovine spermatozoa. Journal of Animal Science, 10(4), 981-987. Walton, A. (1933). Technique of artificial in semination. mp. Bur. Anim. Genet. 56, Iiius – Edinburgh. Warmington, B. G., and Kirton, A. H. (1990). Genetic and non-genetic influences on growth and carcass traits of goats. Small Ruminant Research, 3(2), 147-165.