Animal Reproduction (AR)
Animal Reproduction (AR)

Sperm HSP70: may not be an age-dependent gene but is associated with field fertility in Bali bulls (Bos sondaicus)

Dian Tria Fatmila; Berlin Pandapotan Pardede; Tulus Maulana; Syahruddin Said; Yudi Yudi; Bambang Purwantara

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This study aimed to analyze the characteristics of the HSP70 gene and protein in spermatozoa of Bali bulls of different age groups and to examine its potential as a biomarker determining bull fertility. This study used frozen semen produced from six Bali bulls divided into two groups based on age (≤ 9 years and ≥ 12 years). Parameters of frozen semen quality analyzed included sperm motility and kinetics using computer-assisted semen analysis, sperm morphological defects using Diff-Quick staining, acrosome integrity using FITC-PNA staining, and DNA fragmentation using acridine orange staining. HSP70 gene expression characterization was analyzed using qRT-PCR, and HSP70 protein abundance was analyzed using enzyme immunoassays. Fertility field data were obtained by analyzing the percentage conception rate for each bull based on the artificial insemination service data contained in the Indonesian-integrated system of the National Animal Health Information System (iSIKHNAS). The results showed significant differences (P<0.05) in total and progressive motility, morphological defects of the neck and midpiece, and tail of sperm, and acrosome integrity between the age groups of Bali bulls. HSP70 gene expression and protein abundance showed no significant differences (P>0.05) in different age groups. HSP70 gene expression correlated with fertility rate (P<0.05). Age affected several semen quality parameters but did not affect HSP70 gene expression and protein abundance. The HSP70 gene molecule could be a biomarker that determines the fertility of Bali bulls.


age, Bali bulls, fertility, gene, HSP70, semen quality


Abdulla AK, Rebai T, Al-Delemi DHJ. Protective effects of autologous platelet-rich plasma (PRP) on the cryopreservation outcome in rabbit sperm. Cell Mol Biol. 2022;68(4):113-21. PMid:35988278.

Agarwal A, Selvam MKP, Baskaran S. Proteomic analyses of human sperm cells: understanding the role of protein and molecular pathways affecting male reproductive health. Int J Mol Sci. 2020;21(5):1621. PMid:32120839.

Andrabi SM. Factors affecting the quality of cryopreserved buffalo (Bubalus bubalis) bull spermatozoa. Reprod Domest Anim. 2009;44(3):552-69. PMid:18954384.

Auger J, Jouannet P, Eustache F. Another look at human sperm morphology. Hum Reprod. 2016;31(1):10-23. PMid:26472152.

Bashiri Z, Amidi F, Amiri I, Zandieh Z, Maki CB, Mohammadi F, Amiri S, Koruji M. Male factors: the role of sperm in preimplantation embryo quality. Reprod Sci. 2021;28(7):1788-811. PMid:33140326.

Baumber J, Ball BA, Linfor JJ, Meyers SA. Reactive oxygen species and cryopreservation promote DNA fragmentation in equine spermatozoa. J Androl. 2003;24(4):621-8. PMid:12826702.

Bisht S, Dada R. Oxidative stress: major executioner in disease pathology, role in sperm DNA damage and preventive strategies. Front Biosci (Schol Ed). 2017;9(3):420-47. PMid:28410127.

Carreira JT, Trevizan JT, Carvalho IR, Kipper B, Rodrigues LH, Silva C, Perri SHV, Drevet JR, Koivisto MB. Does sperm quality and DNA integrity differ in cryopreserved semen samples from young, adult, and aged Nellore bulls? Basic Clin Androl. 2017;27(1):12. PMid:28649382.

Cheng Y, Liu S, Zhang Y, Su D, Wang G, Lv C, Zhang Y, Yu H, Hao L, Zhang J. The effect of heat stress on bull sperm quality and related HSPs expression. Anim Biol Leiden Neth. 2016;66(3-4):321-33.

Chenoweth PJ. Genetic sperm defects. Theriogenology. 2005;64(3):457-68. PMid:15993939.

Chianese R, Pierantoni R. Mitochondrial reactive oxygen species (ROS) production alters sperm quality. Antioxidants. 2021;10(1):92. PMid:33440836.

Clancy S, Brown W. Translation: DNA to mRNA to protein. Nature Education. 2008;1(1):101.

Cocchia N, Pasolini MP, Mancini R, Petrazzuolo O, Cristofaro I, Rosapane I, Sica A, Tortora G, Lorizio R, Paraggio G, Mancini A. Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa. Theriogenology. 2011;75(7):1201-10. PMid:21295831.

du Plessis SS, Agarwal A, Mohanty G, van der Linde M. Oxidative phosphorylation versus glycolysis: what fuel do spermatozoa use? Asian J Androl. 2015;17(2):230-5. PMid:25475660.

Egelkrout E, Rajan V, Howard JA. Overproduction of recombinant proteins in plants. Plant Sci. 2012;184:83-101. PMid:22284713.

Fischer D, Neumann D, Wehrend A, Lierz M. Comparison of conventional and computer-assisted semen analysis in cockatiels (Nymphicus hollandicus) and evaluation of different insemination dosages for artificial insemination. Theriogenology. 2014;82(4):613-20. PMid:24985563.

Gangwar C, Mishra AK, Gururaj K, Kumar A, Kharche SD, Saraswat S, Kumar R, Ramachandran N. Semen quality and total microbial load: an association study in important Indian Goat breeds during different seasons. Andrologia. 2021;53(4):e13995. PMid:33629781.

Grzmil P, Boinska D, Kleene KC, Adham I, Schlüter G, Kämper M, Buyandelger B, Meinhardt A, Wolf S, Engel W. Prm3, the fourth gene in the mouse protamine gene cluster, encodes a conserved acidic protein that affects sperm motility. Biol Reprod. 2008;78(6):958-67. PMid:18256328.

Gunes S, Hekim GN, Arslan MA, Asci R. Effects of aging on the male reproductive system. J Assist Reprod Genet. 2016;33(4):441-54. PMid:26867640.

Hardjopranjoto S. Ilmu Kemajiran Pada Ternak. Surabaya: Airlangga University Press; 1995.

Hellstrom WJ, Overstreet JW, Sikka SC, Denne J, Ahuja S, Hoover AM, Sides GD, Cordell WH, Harrison LM, Whitaker JS. Semen and sperm reference ranges for men 45 years of age and older. J Androl. 2006;27(3):421-8. PMid:16452528.

Huang SY, Kuo YH, Lee YP, Tsou HL, Lin EC, Ju CC, Lee WC. Association of heat shock protein 70 with semen quality in boars. Anim Reprod Sci. 2000;63(3-4):231-40. PMid:10989233.

Hung JJ, Cheng TJ, Chang MD, Chen KD, Huang HL, Lai YK. Involvement of heat shock elements and basal transcription elements in the differential induction of the 70-kDa heat shock protein and its cognate by cadmium chloride in 9L rat brain tumor cells. J Cell Biochem. 1998;71(1):21-35.<21::AID-JCB3>3.0.CO;2-3. PMid:9736451.

Indriastuti R, Pardede BP, Gunawan A, Ulum MF, Arifiantini RI, Purwantara B. Sperm transcriptome analysis accurately reveals male fertility potential in livestock. Animals (Basel). 2022;12(21):2955. PMid:36359078.

Kamaruddin M, Kroetsch T, Basrur PK, Hansen PJ, King WA. Immunolocalization of heat shock protein 70 in bovine spermatozoa. Andrologia. 2004;36(5):327-34. PMid:15458553.

Kidd SA, Eskenazi B, Wyrobek AJ. Effects of male age on semen quality and fertility: a review of the literature. Fertil Steril. 2001;75(2):237-48. PMid:11172821.

Kurashova NA, Madaeva IM, Kolesnikova LI. Expression of heat shock proteins HSP70 under oxidative stress. Adv Gerontol. 2019;32(4):502-8. PMid:31800176.

Kusumawati A, Satrio FA, Indriastuti R, Rosyada ZNA, Pardede BP, Agil M, Purwantara B. Sperm head morphology alterations associated with chromatin instability and lack of protamine abundance in frozen-thawed sperm of Indonesian local bulls. Animals (Basel). 2023;13(15):2433. PMid:37570242.

Lemma A. Effect of cryopreservation on sperm quality and fertility. Academia (Caracas). 2011;12:191-216.

Li CJ, Wang D, Zhou X. Sperm proteome and reproductive technologies in mammals. Anim Reprod Sci. 2016;173:1-7. PMid:27576173.

Lopes S, Sun JG, Jurisicova A, Meriano J, Casper RF. Sperm deoxyribonucleic acid fragmentation is increased in poor-quality semen samples and correlates with failed fertilization in intracytoplasmic sperm injection. Fertil Steril. 1998;69(3):528-32. PMid:9531891.

Moein-Vaziri N, Phillips I, Smith S, Almiňana C, Maside C, Gil MA, Roca J, Martinez EA, Holt WV, Pockley AG, Fazeli A. Heat-shock protein A8 restores sperm membrane integrity by increasing plasma membrane fluidity. Reproduction. 2014;147(5):719-32. PMid:24501193.

Mohammed EE, Mosad E, Zahran AM, Hameed DA, Taha EA, Mohamed MA. Acridine orange and flow cytometry: which is better to measure the effect of varicocele on sperm DNA integrity? Adv Urol. 2015;2015:814150. PMid:26681938.

Morrell JM, Nongbua T, Valeanu S, Lima Verde I, Lundstedt-Enkel K, Edman A, Johannisson A. Sperm quality variables as indicators of bull fertility may be breed dependent. Anim Reprod Sci. 2017;185:42-52. PMid:28811063.

Mostek A, Dietrich MA, Słowińska M, Ciereszko A. Cryopreservation of bull semen is associated with carbonylation of sperm proteins. Theriogenology. 2017;92:95-102. PMid:28237350.

Narud B, Khezri A, Nordborg A, Klinkenberg G, Zeremichael TT, Stenseth EB, Heringstad B, Kommisrud E, Myromslien FD. Semen quality parameters including metabolites, sperm production traits, and fertility in young Norwegian Red AI bulls. Livest Sci. 2022;255:2-32.

Özbek M, Hitit M, Kaya A, Jousan FD, Memili E. Sperm functional genome associated with bull fertility. Front Vet Sci. 2021;8:610888. PMid:34250055.

Panner Selvam MK, Agarwal A, Henkel R, Finelli R, Robert KA, Iovine C, Baskaran S. The effect of oxidative and reductive stress on semen parameters and functions of physiologically normal human spermatozoa. Free Radic Biol Med. 2020;152:375-85. PMid:32165282.

Pardede BP, Agil M, Karja NWK, Sumantri C, Supriatna I, Purwantara B. PRM1 gene expression and its protein abundance in frozen-thawed spermatozoa as potential fertility markers in breeding bulls. Vet Sci. 2022;9(3):111. PMid:35324839.

Pardede BP, Agil M, Yudi Y, Supriatna I. Relationship of frozen-thawed semen quality with the fertility rate after being distributed in the Brahman Cross Breeding Program. Vet World. 2020a;13(12):2649-57. PMid:33487983.

Pardede BP, Supriatna I, Yudi Y, Agil M. Decreased bull fertility: age-related changes in sperm motility and DNA fragmentation. E3S Web Conf. 2020b;151:01010.

Pardede BP, Agil M, Supriatna I. Protamine and other proteins in sperm and seminal plasma as molecular markers of bull fertility. Vet World. 2020c;13(3):556-62. PMid:32367964.

Pardede BP, Karja NWK, Said S, Kaiin EM, Agil M, Sumantri C, Purwantara B, Supriatna I. Bovine nucleoprotein transitions genes and protein abundance as valuable markers of sperm motility and the correlation with fertility. Theriogenology. 2024;215:86-94. PMid:38016305.

Pardede BP, Kusumawati A, Pangestu M, Purwantara B. Bovine sperm HSP-70 molecules: a potential cryo-tolerance marker associated with semen quality and fertility rate. Front Vet Sci. 2023;10:1167594. PMid:37621869.

Pardede BP, Maulana T, Kaiin EM, Agil M, Karja NWK, Sumantri C, Supriatna I. The potential of sperm bovine protamine as a protein marker of semen production and quality at the National Artificial Insemination Center of Indonesia. Vet World. 2021;14(9):2473-81. PMid:34840468.

Paul C, Robaire B. Ageing of the male germ line. Nat Rev Urol. 2013;10(4):227-34. PMid:23443014.

Pino V, Sanz A, Valdés N, Crosby J, Mackenna A. The effects of aging on semen parameters and sperm DNA fragmentation. JBRA Assist Reprod. 2020;24(1):82-6. PMid:31692316.

Purwantara B, Noor RR, Andersson G, Rodriguez-Martinez H. Banteng and Bali cattle in Indonesia: status and forecasts. Reprod Domest Anim. 2012;47(s1, Suppl 1):2-6. PMid:22212203.

Rajoriya JS, Prasad JK, Ghosh SK, Perumal P, Kumar A, Kaushal S, Ramteke SS. Studies on effect of different seasons on expression of HSP70 and HSP90 gene in sperm of Tharparkar bull semen. Asian Pac J Reprod. 2014;3(3):192-9.

Razavi S, Hashemi F, Khadivi F, Bakhtiari A, Mokhtarian A, Mirzaei H. Improvement of rat sperm chromatin integrity and spermatogenesis with omega 3 following bleomycin, etoposide and cisplatin treatment. Nutr Cancer. 2021;73(3):514-22. PMid:32321280.

Reddy VS, Yadav B, Yadav CL, Anand M, Swain DK, Kumar D, Kritania D, Madan AK, Kumar J, Yadav S. Effect of sericin supplementation on heat shock protein 70 (HSP70) expression, redox status and post thaw semen quality in goat. Cryobiology. 2018;84:33-9. PMid:30098997.

Rennemeier C, Frambach T, Hennicke F, Dietl J, Staib P. Microbial quorum-sensing molecules induce acrosome loss and cell death in human spermatozoa. Infect Immun. 2009;77(11):4990-7. PMid:19687207.

Rezaei N, Mohammadi M, Mohammadi H, Khalatbari A, Zare Z. Acrosome and chromatin integrity, oxidative stress, and expression of apoptosis-related genes in cryopreserved mouse epididymal spermatozoa treated with L-Carnitine. Cryobiology. 2020;95:171-6. PMid:32220594.

Rosenzweig R, Nillegoda NB, Mayer MP, Bukau B. The Hsp70 chaperone network. Nat Rev Mol Cell Biol. 2019;20(11):665-80. PMid:31253954.

Rosyada ZNA, Pardede BP, Kaiin EM, Gunawan M, Maulana T, Said S, Tumbelaka LITA, Solihin DD, Ulum MF, Purwantara B. A proteomic approach to identifying spermatozoa proteins in Indonesian native Madura bulls. Front Vet Sci. 2023a;10:1287676. PMid:38111731.

Rosyada ZNA, Pardede BP, Kaiin EM, Tumbelaka LITA, Solihin DD, Purwantara B, Ulum MF. Identification of heat shock protein70-2 and protamine-1 mRNA, proteins, and analyses of their association with fertility using frozen-thawed sperm in Madura bulls. Anim Biosci. 2023b;36(12):1796-805. PMid:37402446.

Saito H, Hara K, Kitajima S, Tanemura K. Effect of vitamin E deficiency on spermatogenesis in mice and its similarity to aging. Reprod Toxicol. 2020;98:225-32. PMid:33045311.

Saputra DJ, Ihsan MN, Isnaini N. Korelasi antara lingkar skrotum dengan volume semen, konsentrasi dan motilitas spermatozoa pejantan sapi bali. J Ternak Tropika. 2017;18(2):59-68.

Selvaraju S, Swathi D, Ramya L, Lavanya M, Archana SS, Sivaram M. Orchestrating the expression levels of sperm mRNAs reveals CCDC174 as an important determinant of semen quality and bull fertility. Syst Biol Reprod Med. 2021;67(1):89-101. PMid:33190538.

Silver JT, Noble EG. Regulation of survival gene hsp70. Cell Stress Chaperones. 2012;17(1):1-9. PMid:21874533.

Singh A, Kukreti R, Saso L, Kukreti S. Oxidative stress: a key modulator in neurodegenerative diseases. Molecules. 2019;24(8):1583. PMid:31013638.

Somashekar L, Selvaraju S, Parthipan S, Patil SK, Binsila BK, Venkataswamy MM, Karthik Bhat S, Ravindra JP. Comparative sperm protein profiling in bulls differing in fertility and identification of phosphatidylethanolamine-binding protein 4, a potential fertility marker. Andrology. 2017;5(5):1032-51. PMid:28859251.

Spinaci M, Volpe S, Bernardini C, de Ambrogi M, Tamanini C, Seren E, Galeati G. Sperm sorting procedure induces a redistribution of Hsp70 but not Hsp60 and Hsp90 in boar spermatozoa. J Androl. 2006;27(6):899-907. PMid:16870948.

Tsai YR, Lan KC, Kung FT, Lin PY, Chiang HJ, Lin YJ, Huang FJ. The effect of advanced paternal age on the outcomes of assisted reproductive techniques among patients with azoospermia using cryopreserved testicular spermatozoa. Taiwan J Obstet Gynecol. 2013;52(3):351-5. PMid:24075372.

Tuset VM, Dietrich GJ, Wojtczak M, Słowińska M, de Monserrat J, Ciereszko A. Comparison of three staining techniques for the morphometric study of rainbow trout (Oncorhynchus mykiss) spermatozoa. Theriogenology. 2008;69(8):1033-8. PMid:18359066.

Varghese T, Divyashree BC, Roy SC, Roy KS. Loss of heat shock protein 70 from apical region of buffalo (Bubalus bubalis) sperm head after freezing and thawing. Theriogenology. 2016;85(5):828-34. PMid:26607876.

Vihinen M. Types and effects of protein variations. Hum Genet. 2015;134(4):405-21. PMid:25616435.

Wang H, Wan H, Li X, Liu W, Chen Q, Wang Y, Yang L, Tang H, Zhang X, Duan E, Zhao X, Gao F, Li W. Atg7 is required for acrosome biogenesis during spermatogenesis in mice. Cell Res. 2014;24(7):852-69. PMid:24853953.

Wang X, Li T, Yin D, Chen N, An X, Zhao X, Ma Y. Distinct expression and localization patterns of HSP70 in developmental reproductive organs of rams. Gene. 2020;760:145029. PMid:32758578.

Yadav HP, Kumar A, Shah N, Chauhan DS, Lone SA, Swain DK, Saxena A. Effect of cholesterol-loaded cyclodextrin on membrane and acrosome status of Hariana bull sperm during cryopreservation. Cryo Lett. 2018;39(6):386-90. PMid:30963156.

Zainudin M, Ihsan MN, Suyadi S. Efisiensi reproduksi sapi perah PFH pada berbagai umur di cv. milkindo berka abadi desa tegalsari kecamatan kepanjen kabupaten malang. JIIP. 2014;24(3):32-7.

Zhang XG, Hong JY, Yan GJ, Wang YF, Li QW, Hu JH. Association of heat shock protein 70 with motility of frozen-thawed sperm in bulls. Czech J Anim Sci. 2015a;60(6):256-62.

Zhang XG, Hu S, Han C, Zhu QC, Yan GJ, Hu JH. Association of heat shock protein 90 with motility of post-thawed sperm in bulls. Cryobiology. 2015b;70(2):164-9. PMid:25578982.

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