Abstract

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.

Keywords:
age; Bali bulls; fertility; gene; HSP70; semen quality

Introduction

Bali cattle (Bos sondaicus) are a native breed of Indonesian beef cattle, domesticated from wild banteng (Bos javanicus) in Bali and Java for hundreds of years. Bali cattle have various advantages, such as adaptability to different environmental conditions, unfavorable feed, and a reasonably high carcass percentage, reaching around 51% (Saputra et al., 2017Saputra 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. http://dx.doi.org/10.21776/ub.jtapro.2017.018.02.9.
http://dx.doi.org/10.21776/ub.jtapro.201... ). Besides having good productivity, Bali cattle are also reported to have good reproductive performance and fertility (Purwantara et al., 2012Purwantara 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. http://dx.doi.org/10.1111/j.1439-0531.2011.01956.x. PMid:22212203.
http://dx.doi.org/10.1111/j.1439-0531.20... ). The various advantages of Bali cattle have made this local cattle breed one of the superior beef cattle alternatives to meet the increasing demand for beef in Indonesia, so an increase in the population and genetic quality of these local cattle is urgently needed. Artificial Insemination (AI) is an assisted reproductive technology that can improve livestock's genetic quality and productivity. AI generally uses frozen semen produced through cryopreservation and from superior bulls, including Bali bulls. Frozen semen used for AI is categorized as of good quality if it has progressive motility of more than 40% (Pardede et al., 2024Pardede 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. http://dx.doi.org/10.1016/j.theriogenology.2023.11.015. PMid:38016305.
http://dx.doi.org/10.1016/j.theriogenolo... ), the acrosome is intact, not-fragmented DNA, and it contains sperm with abnormal morphology <15% so that they can produce optimal fertility levels (Rezaei et al., 2020Rezaei 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. http://dx.doi.org/10.1016/j.cryobiol.2020.03.006. PMid:32220594.
http://dx.doi.org/10.1016/j.cryobiol.202... ). Pardede et al. (2020a)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. http://dx.doi.org/10.14202/vetworld.2020.2649-2657. PMid:33487983.
http://dx.doi.org/10.14202/vetworld.2020... reported that semen quality is closely related to bull fertility. Furthermore, it was reported that apart from semen quality, increasing age can affect semen quality and have an impact on decreasing the fertility of bulls (Pardede et al., 2020bPardede 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. http://dx.doi.org/10.1051/e3sconf/202015101010.
http://dx.doi.org/10.1051/e3sconf/202015... ).

Increasing age is one of the factors that can affect the metabolic system in the body that cannot be avoided. Aging can cause a decrease in semen quality (Tsai et al., 2013Tsai 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. http://dx.doi.org/10.1016/j.tjog.2013.06.001. PMid:24075372.
http://dx.doi.org/10.1016/j.tjog.2013.06... ) and impact decreasing fertility (Kidd et al., 2001Kidd 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. http://dx.doi.org/10.1016/S0015-0282(00)01679-4. PMid:11172821.
http://dx.doi.org/10.1016/S0015-0282(00)... ). Various species have reported that age affects fertility (Pardede et al., 2020bPardede 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. http://dx.doi.org/10.1051/e3sconf/202015101010.
http://dx.doi.org/10.1051/e3sconf/202015... ; Gangwar et al., 2021Gangwar 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. http://dx.doi.org/10.1111/and.13995. PMid:33629781.
http://dx.doi.org/10.1111/and.13995... ). Aging can cause a physiological decline in the reproductive organs that can cause disturbances in spermatogenesis. Gonadotropin levels increase, and testosterone levels decrease with aging, decreasing the quantity of Leydig, Sertoli, and germ cells. It reduces reproductive performance, including semen quality and fertility of bulls (Pino et al., 2020Pino 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. http://dx.doi.org/10.5935/1518-0557.20190058. PMid:31692316.
http://dx.doi.org/10.5935/1518-0557.2019... ). In addition, aging also makes cells vulnerable to oxidative stress (ROS) due to mitochondrial dysfunction (Singh et al., 2019Singh A, Kukreti R, Saso L, Kukreti S. Oxidative stress: a key modulator in neurodegenerative diseases. Molecules. 2019;24(8):1583. http://dx.doi.org/10.3390/molecules24081583. PMid:31013638.
http://dx.doi.org/10.3390/molecules24081... ). Reactive oxygen species (ROS), one of the excess oxidant products, can initiate cell damage, leading to apoptosis (Singh et al., 2019Singh A, Kukreti R, Saso L, Kukreti S. Oxidative stress: a key modulator in neurodegenerative diseases. Molecules. 2019;24(8):1583. http://dx.doi.org/10.3390/molecules24081583. PMid:31013638.
http://dx.doi.org/10.3390/molecules24081... ) and cause a decrease in semen quality, one of the essential factors that can affect bull fertility.

Although it is one of the essential factors that can affect the fertility of bulls, recent studies have shown that good semen quality does not guarantee optimal fertility in the field. Pardede et al. (2022)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. http://dx.doi.org/10.3390/vetsci9030111. PMid:35324839.
http://dx.doi.org/10.3390/vetsci9030111... reported that although the quality of semen met specific requirements and was considered to be of good quality, three out of six bulls in the field showed a low fertility rate (<50%). Recent research has found various gene and protein molecules in spermatozoa that play an essential role in the reproductive process of bulls, mainly the physiological functions of sperm oocytes, which are believed to be able to predict bull fertility accurately (Özbek et al., 2021Özbek M, Hitit M, Kaya A, Jousan FD, Memili E. Sperm functional genome associated with bull fertility. Front Vet Sci. 2021;8:610888. http://dx.doi.org/10.3389/fvets.2021.610888. PMid:34250055.
http://dx.doi.org/10.3389/fvets.2021.610... ; Li et al., 2016Li CJ, Wang D, Zhou X. Sperm proteome and reproductive technologies in mammals. Anim Reprod Sci. 2016;173:1-7. http://dx.doi.org/10.1016/j.anireprosci.2016.08.008. PMid:27576173.
http://dx.doi.org/10.1016/j.anireprosci.... ; Pardede et al., 2024Pardede 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. http://dx.doi.org/10.1016/j.theriogenology.2023.11.015. PMid:38016305.
http://dx.doi.org/10.1016/j.theriogenolo... ; Rosyada et al., 2023aRosyada 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. http://dx.doi.org/10.3389/fvets.2023.1287676. PMid:38111731.
http://dx.doi.org/10.3389/fvets.2023.128... ). Furthermore, this is due to the involvement of more complex molecular mechanisms in spermatozoa, including the loss of spermatozoa surface protein required for fertilization (Li et al., 2016Li CJ, Wang D, Zhou X. Sperm proteome and reproductive technologies in mammals. Anim Reprod Sci. 2016;173:1-7. http://dx.doi.org/10.1016/j.anireprosci.2016.08.008. PMid:27576173.
http://dx.doi.org/10.1016/j.anireprosci.... ).

Heat shock protein 70 (HSP70) is a molecule in sperm with a molecular weight of 70 kDa, which has a role in maintaining homeostasis from changes in temperature and oxidative stress (Rosenzweig et al., 2019Rosenzweig R, Nillegoda NB, Mayer MP, Bukau B. The Hsp70 chaperone network. Nat Rev Mol Cell Biol. 2019;20(11):665-80. http://dx.doi.org/10.1038/s41580-019-0133-3. PMid:31253954.
http://dx.doi.org/10.1038/s41580-019-013... ; Cheng et al., 2016Cheng 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. http://dx.doi.org/10.1163/15707563-00002507.
http://dx.doi.org/10.1163/15707563-00002... ). Agarwal et al. (2020)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. http://dx.doi.org/10.3390/ijms21051621. PMid:32120839.
http://dx.doi.org/10.3390/ijms21051621... reported that HSP70 in bull spermatozoa is associated with motility, capacitation, and spermatozoa-oocyte interactions. The abundance of HSP70 protein in several bulls, such as Holstein Friesian bulls (Somashekar et al., 2017Somashekar 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. http://dx.doi.org/10.1111/andr.12404. PMid:28859251.
http://dx.doi.org/10.1111/andr.12404... ), Tharparkar (Rajoriya et al., 2014Rajoriya 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. http://dx.doi.org/10.1016/S2305-0500(14)60025-7.
http://dx.doi.org/10.1016/S2305-0500(14)... ), and Simmental (Cheng et al., 2016Cheng 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. http://dx.doi.org/10.1163/15707563-00002507.
http://dx.doi.org/10.1163/15707563-00002... ) was found in spermatozoa. It was characterized through a proteomic technology approach. Furthermore, Bashiri et al. (2021)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. http://dx.doi.org/10.1007/s43032-020-00334-z. PMid:33140326.
http://dx.doi.org/10.1007/s43032-020-003... stated that the expression of the HSP gene in spermatozoa contributes to the early development of the embryo, and changes in its expression pattern can cause pregnancy failure and reduce fertility. This study aims to analyze the characteristics of the HSP70 gene and protein in spermatozoa of Bali bulls in different age groups and to examine its potential as a biomarker determining bull fertility, especially in Bali bulls, which has never been reported before.

Methods

Ethical approval

The sample used is a commercial product and is not directly related to the bulls used in the study. Every procedure, from storing fresh semen to turning it into frozen semen, is carried out according to the protocol set at the Bali AI center and has complied with every animal welfare principle.

Experimental animals

The sample used in this study was frozen semen produced at the Regional Artificial Insemination Center, Bali Province, Indonesia. This study only used frozen semen, a commercial product from the AI center ready to be distributed in the field, as the primary sample and did not directly involve bulls. Furthermore, the entire semen collection process was carried out using an artificial vagina for the same period (without seasonal differences), frozen using the same extender. To eliminate any potential for variation in the samples, the bulls were kept in the same environment regarding feeding and handling management. Frozen semen produced from six Bali bulls divided into two groups based on age (≤ 9 years and ≥ 12 years) was used in this study for further analysis. This grouping is based on Zainudin et al. (2014)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., which state that productive bulls are less than ten years old. At the age of more than ten years, there is a decrease in reproductive ability physiologically and hormonally.

Frozen semen quality

Frozen sperm from each bull was defrosted in a 37°C water bath for 30 seconds before being placed in a microtube for further analysis. The parameters of semen quality tested in this study were sperm motility and kinetics, morphology, acrosome integrity, and DNA fragmentation. The stages of this study used five biological repeats for each of the tested semen quality parameters. Sperm motility and kinetic analysis were carried out using computer-assisted semen analysis (CASA) with the Sperm-Vision Program (Minitüb, Tiefenbach, Germany), which was linked to Carl Zeiss Microimaging GmbH (Gottingen, Germany) and equipped with a warm stage at 38°C. The parameters analyzed were total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight-line velocity (VSL), average path velocity (VAP), linearity (LIN), and straightness (STR) (Fischer et al., 2014Fischer 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. http://dx.doi.org/10.1016/j.theriogenology.2014.05.023. PMid:24985563.
http://dx.doi.org/10.1016/j.theriogenolo... ).

Sperm morphological defects were analyzed using the commercial Diff-Quick Staining Kit (Aurora Scientific, Indonesia), which refers to the method of Tuset et al. (2008)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. http://dx.doi.org/10.1016/j.theriogenology.2008.01.012. PMid:18359066.
http://dx.doi.org/10.1016/j.theriogenolo... . Sperm morphological defects are classified into three groups: head defects, neck and mid-piece defects, and tail defects (Chenoweth, 2005Chenoweth PJ. Genetic sperm defects. Theriogenology. 2005;64(3):457-68. http://dx.doi.org/10.1016/j.theriogenology.2005.05.005. PMid:15993939.
http://dx.doi.org/10.1016/j.theriogenolo... ). Analysis of the integrity of the sperm acrosome in this study was carried out using the FITC-PNA staining method, according to Cocchia et al. (2011)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. http://dx.doi.org/10.1016/j.theriogenology.2010.11.031. PMid:21295831.
http://dx.doi.org/10.1016/j.theriogenolo... . The DNA damage of spermatozoa was examined using acridine orange (AO) staining, as described by Pardede et al. (2021)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. http://dx.doi.org/10.14202/vetworld.2021.2473-2481. PMid:34840468.
http://dx.doi.org/10.14202/vetworld.2021... .

HSP70 gene expression analysis

Semen samples thawed in a water bath at 37°C for 30 seconds were then placed in a microtube. Then, RNA was isolated and extracted from each semen sample using the DirectZol RNA kit (Zymo Research, USA), and reverse transcription and cDNA synthesis was performed using the SensiFAS™ cDNA Synthesis kit (Bioline Ltd, UK). Then, the master mix consisting of the SensiFAST™ SYBR® No-ROX kit (Bioline Ltd, UK), forward and reverse primers, nuclease-free water, and cDNA was added to the PCR plate. The primer sequences used for HSP70 gene expression analysis (NM_174344.1) were forward 5'-TTGGGGACAAGTCAGAGAATG-3' and reverse 5'-ATCGTGGGGTTCCTTTTGATG-3'. The HSP70 primer was obtained from literature studies where this primer has been applied and used on local bull breeds, such as Qinchuan (Bos taurus) (Zhang et al., 2015aZhang 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. http://dx.doi.org/10.17221/8239-CJAS.
http://dx.doi.org/10.17221/8239-CJAS... ), Madura (Bos javanicus x Bos indicus) (Rosyada et al., 2023bRosyada 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. http://dx.doi.org/10.5713/ab.23.0142. PMid:37402446.
http://dx.doi.org/10.5713/ab.23.0142... ), and Bali (Bos sondaicus) (Pardede et al., 2023Pardede 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. http://dx.doi.org/10.3389/fvets.2023.1167594. PMid:37621869.
http://dx.doi.org/10.3389/fvets.2023.116... ) bulls. PPIA (NM_178320.2) was used as an endogenous control, with the primary sequence being forward 5'-ATGCTGGCCCCAACACAA-3' and reverse 5'-CCCTCTCACCTTGCCAAA-3'. Gene expression analysis was performed using the quantitative reverse transcription PCR (qRT-PCR) method, using the Bio-Rad CFX OPUS 96 Real-Time PCR System. This quantification analysis obtained the cycle threshold (C_T) value representing the nucleic acid copy number. The relative quantification value of HSP70 gene expression was calculated using the 2^-ΔΔC_t formula, which compared the C_T value of the target gene with PPIA.

HSP70 protein abundance analysis

The frozen sperm sample was thawed in a 37°C water bath for 30 seconds before being centrifuged at 1000 x g for 20 minutes. HSP70 protein abundance was analyzed using enzyme immunoassays (EIA) according to the protocol instructions for use on the bovine HSP70 kit (Cat No. MBS7606199, MyBioSource.com). The absorbance value will be determined using an EIA reader at 450 nm. The abundance of HSP70 protein in a sample can be determined by comparing its value with the standard curve (Mostek et al. 2017Mostek A, Dietrich MA, Słowińska M, Ciereszko A. Cryopreservation of bull semen is associated with carbonylation of sperm proteins. Theriogenology. 2017;92:95-102. http://dx.doi.org/10.1016/j.theriogenology.2017.01.011. PMid:28237350.
http://dx.doi.org/10.1016/j.theriogenolo... ).

Field fertility analysis

The field fertility assessment is determined by the value of the first service conception rate of each bull based on the success of AI. The data are in the form of an AI application using frozen semen straws from bulls used in the study and the results of pregnancy examinations for each inseminated female cow. More than 1,000 data on the success of AI over the last two years were used in the study to analyze the percentage of first service conception rate in each bull using the Pardede et al. (2020a)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. http://dx.doi.org/10.14202/vetworld.2020.2649-2657. PMid:33487983.
http://dx.doi.org/10.14202/vetworld.2020... technique. The first service conception rate was defined as the number of pregnant cows (80–100 days after the first AI service) to all inseminated cows. However, the AI success data used is secondary data reported by AI officers in the field, which is contained and recorded in the Indonesian-integrated National Animal Health Information System (iSIKHNAS) and does not directly involve female cows. The %fertility rate analysis results for each bull sequentially were 74.60%, 78.09%, 75.13% (≤ 9 years bulls), and 73.43%, 74.67%, and 74.50% (≥ 12 years bulls). The analysis results were then used to predict the %field fertility rate for each bull in the study.

Statistical analysis

The independent t-test was used to analyze data on differences in semen quality, gene expression and protein of HSP70, and field fertility between age groups. The relationship between gene expression and protein of HSP70 with semen quality and field fertility was analyzed using the Pearson correlation test. The correlation pattern between gene expression and protein abundance of HSP70 with field fertility was determined using the Scatter Plot linearity test. Data was analyzed using SPSS program version 25.0 (IBM, Armonk, NY, USA). Data are presented in the mean ± standard error.

Results

The findings revealed a significant variation (P<0.05) in total (63.81±0.84% vs. 57.91±1.50%) and progressive motility (57.89±1.05% vs. 52.14±1.70%) between age groups of Bali bulls (Figure 1). Kinetic parameters of sperm cells, such as VCL, VSL, VAP, STR, and LIN, revealed no significant difference (P>0.05) among Bali bull ages (Table 1).

The results showed that there were significant differences (P<0.05) in the sperm morphological defects, the defects in the neck and midpiece of the sperm (1.01±0.28% vs. 2.10±0.26%) and the tail of the sperm (7.56±0.28% vs. 9.18±0.38%) between the age groups of Bali bulls (Figure 2). At the same time, there was no significant difference (P>0.05) in the head defects (P>0.05) between the age groups (Figure 2).

This study found a significant difference (P<0.05) in sperm acrosome integrity analyzed using the FITC-PNA method (Figure 3) between the age groups of Bali bulls (85.14±0.82% vs. 82.01±1.16%) (Figure 4). Sperm DNA fragmentation assessed using the AO staining method (Figure 3) in this study was not significantly different (P>0.05) between age groups of bulls (Table 2). In addition, the percentage of field fertility between the age groups of bulls did not differ significantly (P>0.05) (Table 2).

HSP70 gene expression (4.42±0.62 vs. 2.81±0.26) and its protein abundance (0.0081±0.00021 ng/mL vs. 0.0106±0.00085 ng/mL) in this study showed no significant difference (P>0.05) between the age groups of Bali bulls (Figure 5). The results of the correlation analysis between HSP70 gene expression and semen quality are shown in Table 3. In contrast, the results of the correlation analysis between HSP70 protein abundance and semen quality are shown in Table 4. The results showed that HSP70 gene expression positively correlated with field fertility (P<0.05) (Table 4). The results of the linearity test using the Scatter Plot curve show that only HSP70 gene expression has a linear and positive relationship with field fertility (Figure 6).

Discussion

Fertility is a multifactorial phenomenon influenced by many factors, including the bull factor (Morrell et al., 2017Morrell 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. http://dx.doi.org/10.1016/j.anireprosci.2017.08.001. PMid:28811063.
http://dx.doi.org/10.1016/j.anireprosci.... ). A decrease in bull fertility can cause substantial economic losses in the livestock sector (Özbek et al., 2021Özbek M, Hitit M, Kaya A, Jousan FD, Memili E. Sperm functional genome associated with bull fertility. Front Vet Sci. 2021;8:610888. http://dx.doi.org/10.3389/fvets.2021.610888. PMid:34250055.
http://dx.doi.org/10.3389/fvets.2021.610... ). Selvaraju et al. (2021)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. http://dx.doi.org/10.1080/19396368.2020.1836286. PMid:33190538.
http://dx.doi.org/10.1080/19396368.2020.... stated that several factors, including semen quality, influence bull fertility. In addition to semen quality, bull fertility and reproductive capacity are also affected by age (Narud et al., 2022Narud 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. http://dx.doi.org/10.1016/j.livsci.2021.104803.
http://dx.doi.org/10.1016/j.livsci.2021.... ; Gunes et al., 2016Gunes S, Hekim GN, Arslan MA, Asci R. Effects of aging on the male reproductive system. J Assist Reprod Genet. 2016;33(4):441-54. http://dx.doi.org/10.1007/s10815-016-0663-y. PMid:26867640.
http://dx.doi.org/10.1007/s10815-016-066... ), which can be seen from a decrease in semen quality. As found in this study, there was a decrease in several parameters of semen quality in the group of bulls over 12 years of age. Overall, the motility of spermatozoa in this study was far above the minimum standard (>40%) for frozen semen quality values determined based on the Indonesian National Standard. The field fertility analysis for each Bali bull in this study also showed promising results. Hardjopranjoto (1995)Hardjopranjoto S. Ilmu Kemajiran Pada Ternak. Surabaya: Airlangga University Press; 1995. stated that the success of artificial insemination, as measured by the level of fertility in the field, is categorized as good if it reaches a value of 65-75%.

Various studies have reported the effect of age on decreasing semen quality and bull fertility (Carreira et al., 2017Carreira 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. http://dx.doi.org/10.1186/s12610-017-0056-9. PMid:28649382.
http://dx.doi.org/10.1186/s12610-017-005... ). Gunes et al. (2016)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. http://dx.doi.org/10.1007/s10815-016-0663-y. PMid:26867640.
http://dx.doi.org/10.1007/s10815-016-066... reported that aging is correlated with increased oxidative stress, which causes an increase in lipid peroxidation and the formation of ROS in mitochondria. Singh et al. (2019)Singh A, Kukreti R, Saso L, Kukreti S. Oxidative stress: a key modulator in neurodegenerative diseases. Molecules. 2019;24(8):1583. http://dx.doi.org/10.3390/molecules24081583. PMid:31013638.
http://dx.doi.org/10.3390/molecules24081... also stated that oxygen in mitochondria is essential in oxidative phosphorylation, which involves glucose breakdown and ATP formation. ROS plays a critical role in male fertility (du Plessis et al., 2015du 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. http://dx.doi.org/10.4103/1008-682X.135123. PMid:25475660.
http://dx.doi.org/10.4103/1008-682X.1351... ); low ROS levels are needed for several physiological processes of spermatozoa, but excessive amounts can cause male infertility (Bisht and Dada, 2017Bisht 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. http://dx.doi.org/10.2741/s495. PMid:28410127.
http://dx.doi.org/10.2741/s495... ). ROS concentrations increase with age, along with a decrease in antioxidant levels (Paul and Robaire, 2013Paul C, Robaire B. Ageing of the male germ line. Nat Rev Urol. 2013;10(4):227-34. http://dx.doi.org/10.1038/nrurol.2013.18. PMid:23443014.
http://dx.doi.org/10.1038/nrurol.2013.18... ). Excessive ROS production can cause damage to sperm cells (Andrabi, 2009Andrabi SM. Factors affecting the quality of cryopreserved buffalo (Bubalus bubalis) bull spermatozoa. Reprod Domest Anim. 2009;44(3):552-69. http://dx.doi.org/10.1111/j.1439-0531.2008.01240.x. PMid:18954384.
http://dx.doi.org/10.1111/j.1439-0531.20... ). Furthermore, aging causes histological changes in the testes, such as alteration in the morphology of germ cells, Sertoli cells, peritubular testes, and Leydig cells (Gunes et al., 2016Gunes S, Hekim GN, Arslan MA, Asci R. Effects of aging on the male reproductive system. J Assist Reprod Genet. 2016;33(4):441-54. http://dx.doi.org/10.1007/s10815-016-0663-y. PMid:26867640.
http://dx.doi.org/10.1007/s10815-016-066... ). ROS are widely known in spermatozoa as second messengers in important cellular events related to the fertilization process, such as acrosome reaction, hyperactivation, and spermatozoa-oocyte fusion, so the physiological amount of ROS is essential to maintain cell balance.

Besides impacting increasing oxidative stress, aging is also reported to be associated with decreased tryptophan levels in seminal plasma. Tryptophan is an amino acid that plays a role in 5-hydroxytryptamine synthesis, which supports the acrosome reaction and regulates sperm motility and tyrosine phosphorylation activity (Narud et al., 2022Narud 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. http://dx.doi.org/10.1016/j.livsci.2021.104803.
http://dx.doi.org/10.1016/j.livsci.2021.... ). Chianese and Pierantoni (2021)Chianese R, Pierantoni R. Mitochondrial reactive oxygen species (ROS) production alters sperm quality. Antioxidants. 2021;10(1):92. http://dx.doi.org/10.3390/antiox10010092. PMid:33440836.
http://dx.doi.org/10.3390/antiox10010092... also reported that aging correlates with poor semen quality, one of which is motility. The restriction of mitochondrial membrane potential, which disrupts the process of forming ATP, which is required for the pattern of movement of spermatozoa, causes a decrease in sperm motility. Previous research has also found that aging reduces sperm motility and kinetic parameters (Hellstrom et al., 2006Hellstrom 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. http://dx.doi.org/10.2164/jandrol.05156. PMid:16452528.
http://dx.doi.org/10.2164/jandrol.05156... ). It is the same as seen in the study, in which there was a significant decrease (P<0.05) in total and progressive spermatozoa motility in bulls over 12 years of age. Nevertheless, there was no statistically significant difference in the kinetic parameters of spermatozoa between bull age groups.

Sperm morphological defects are influenced by various factors, including environmental and genetic (Chenoweth, 2005Chenoweth PJ. Genetic sperm defects. Theriogenology. 2005;64(3):457-68. http://dx.doi.org/10.1016/j.theriogenology.2005.05.005. PMid:15993939.
http://dx.doi.org/10.1016/j.theriogenolo... ; Auger et al., 2016Auger J, Jouannet P, Eustache F. Another look at human sperm morphology. Hum Reprod. 2016;31(1):10-23. http://dx.doi.org/10.1093/humrep/dev251. PMid:26472152.
http://dx.doi.org/10.1093/humrep/dev251... ). Saito et al. (2020)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. http://dx.doi.org/10.1016/j.reprotox.2020.10.003. PMid:33045311.
http://dx.doi.org/10.1016/j.reprotox.202... reported that aging could cause an increase in sperm morphological defects. The high percentage of sperm morphological defects can cause fertilization failure (Lopes et al., 1998Lopes 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. http://dx.doi.org/10.1016/S0015-0282(97)00536-0. PMid:9531891.
http://dx.doi.org/10.1016/S0015-0282(97)... ). In this study, sperm neck, mid-piece, and tail defects differed significantly (P<0.05) between the age groups. These two morphological defects of the sperm are parts of the sperm that are related to the function of the movement of the sperm, especially the mid-piece, which is where the mitochondria of the cells are located (Chenoweth, 2005Chenoweth PJ. Genetic sperm defects. Theriogenology. 2005;64(3):457-68. http://dx.doi.org/10.1016/j.theriogenology.2005.05.005. PMid:15993939.
http://dx.doi.org/10.1016/j.theriogenolo... ). This finding follows the percentage of total and progressive motility in this research, revealing a drop in quality in the bulls over 12. Chianese and Pierantoni (2021)Chianese R, Pierantoni R. Mitochondrial reactive oxygen species (ROS) production alters sperm quality. Antioxidants. 2021;10(1):92. http://dx.doi.org/10.3390/antiox10010092. PMid:33440836.
http://dx.doi.org/10.3390/antiox10010092... showed that mitochondria are organelles in cells that are the site for ATP production, which produces the energy needed for sperm to be able to move progressively motile until they reach the site of fertilization.

In contrast, there was no substantial difference in sperm head defects between the age groups (P>0.05). Chenoweth (2005)Chenoweth PJ. Genetic sperm defects. Theriogenology. 2005;64(3):457-68. http://dx.doi.org/10.1016/j.theriogenology.2005.05.005. PMid:15993939.
http://dx.doi.org/10.1016/j.theriogenolo... , Carreira et al. (2017)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. http://dx.doi.org/10.1186/s12610-017-0056-9. PMid:28649382.
http://dx.doi.org/10.1186/s12610-017-005... , and Kusumawati et al. (2023)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. http://dx.doi.org/10.3390/ani13152433. PMid:37570242.
http://dx.doi.org/10.3390/ani13152433... reported that sperm head defects, which are significant abnormalities in sperm, are generally caused by various things, such as genetic factors and DNA condensation. High sperm head defects were also reported to correlate with decreased bull fertility (Lemma, 2011Lemma A. Effect of cryopreservation on sperm quality and fertility. Academia (Caracas). 2011;12:191-216.). No differences were found in the abnormalities of the sperm heads in this study, possibly due to the condition of the sperm DNA, which was also in good condition. The findings of this study also show that the sperm DNA fragmentation values between ages were not significantly different (P<0.05). In addition, it can be seen that the fertility rate of bulls in this study is still relatively good.

Sperm DNA integrity is essential for the transmission of the genetic code. It is considered a potential marker of male fertility, so high numbers of fragmented spermatozoa will significantly cause fertilization failure and delay embryo development (Mohammed et al., 2015Mohammed 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. http://dx.doi.org/10.1155/2015/814150. PMid:26681938.
http://dx.doi.org/10.1155/2015/814150... ). There wasn't a substantial difference in DNA fragmentation values (P<0.05) between the age groups in this study. It differs from the findings of Carreira et al. (2017)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. http://dx.doi.org/10.1186/s12610-017-0056-9. PMid:28649382.
http://dx.doi.org/10.1186/s12610-017-005... and Gunes et al. (2016)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. http://dx.doi.org/10.1007/s10815-016-0663-y. PMid:26867640.
http://dx.doi.org/10.1007/s10815-016-066... , who found a link between age and DNA fragmentation. Pardede et al. (2020b)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. http://dx.doi.org/10.1051/e3sconf/202015101010.
http://dx.doi.org/10.1051/e3sconf/202015... also reported an effect of age on increasing DNA fragmentation in bulls, although overall, the fragmentation value was still relatively good, which was less than 15%. High sperm DNA fragmentation is often associated with excess ROS production due to cold shock during sperm cryopreservation (Baumber et al., 2003Baumber 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. http://dx.doi.org/10.1002/j.1939-4640.2003.tb02714.x. PMid:12826702.
http://dx.doi.org/10.1002/j.1939-4640.20... ). Oxidative stress, due to an imbalance in ROS production, can cause DNA fragmentation and affect fertility (Razavi et al., 2021Razavi 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. http://dx.doi.org/10.1080/01635581.2020.1757128. PMid:32321280.
http://dx.doi.org/10.1080/01635581.2020.... ). Increased levels of ROS cause harmful effects at the molecular level of sperm because it results in peroxidation of sperm membrane lipids, loss of protein function, and occurrence of DNA fragmentation, which negatively impacts the normal function of sperm (Panner Selvam et al., 2020Panner 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. http://dx.doi.org/10.1016/j.freeradbiomed.2020.03.008. PMid:32165282.
http://dx.doi.org/10.1016/j.freeradbiome... ). In addition, Pardede et al. (2021)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. http://dx.doi.org/10.14202/vetworld.2021.2473-2481. PMid:34840468.
http://dx.doi.org/10.14202/vetworld.2021... also reported an abnormality in the protamine composition in the sperm nucleus, which resulted in high sperm DNA fragmentation. Protamine is the main protein in the core of mature sperm, which wraps and protects the sperm DNA, which, if a disturbance occurs, will have an impact on reducing bull fertility (Pardede et al., 2020cPardede 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. http://dx.doi.org/10.14202/vetworld.2020.556-562. PMid:32367964.
http://dx.doi.org/10.14202/vetworld.2020... ).

Failure of the acrosome reaction process due to damage to the acrosome cap of sperm is one of the fundamental causes that cause infertility in bulls (Rennemeier et al., 2009Rennemeier 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. http://dx.doi.org/10.1128/IAI.00586-09. PMid:19687207.
http://dx.doi.org/10.1128/IAI.00586-09... ). The integrity of the sperm membrane and acrosome is critical in fertilization (Yadav et al., 2018Yadav 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.). The acrosome is a distinct membranous organelle found in the sperm nucleus's anterior region, which functions in the process of penetration of sperm in the zona pellucida during fertilization (Wang et al., 2014Wang 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. http://dx.doi.org/10.1038/cr.2014.70. PMid:24853953.
http://dx.doi.org/10.1038/cr.2014.70... ). The proportion of acrosome integrity differs substantially (P<0.05) between ages, according to the findings. It follows what Carreira et al. (2017)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. http://dx.doi.org/10.1186/s12610-017-0056-9. PMid:28649382.
http://dx.doi.org/10.1186/s12610-017-005... reported that aging would cause the acrosome in sperm cells to be more susceptible to various damages. This is because aging is associated with decreased antioxidant protective activity due to the cryopreservation process, which also results in a considerable reduction in glutathione peroxidase and superoxidase dismutase (Carreira et al., 2017Carreira 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. http://dx.doi.org/10.1186/s12610-017-0056-9. PMid:28649382.
http://dx.doi.org/10.1186/s12610-017-005... ). In addition, previous studies also reported another effect of the cryopreservation process, particularly as another impact of cold shock on semen quality, namely a decrease in heat shock protein (Huang et al., 2000Huang 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. http://dx.doi.org/10.1016/S0378-4320(00)00175-5. PMid:10989233.
http://dx.doi.org/10.1016/S0378-4320(00)... ; Zhang et al., 2015bZhang 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. http://dx.doi.org/10.1016/j.cryobiol.2014.12.010. PMid:25578982.
http://dx.doi.org/10.1016/j.cryobiol.201... ; Varghese et al., 2016Varghese 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. http://dx.doi.org/10.1016/j.theriogenology.2015.10.029. PMid:26607876.
http://dx.doi.org/10.1016/j.theriogenolo... ).

Cold shock is one of the effects of the cryopreservation phase that has been linked. The decrease in heat shock proteins, including HSP70, increased ROS concentrations and oxidative damage to intracellular enzymes (Kurashova et al., 2019Kurashova NA, Madaeva IM, Kolesnikova LI. Expression of heat shock proteins HSP70 under oxidative stress. Adv Gerontol. 2019;32(4):502-8. PMid:31800176.). HSP70 is a chaperone molecule usually induced during stress on cells (Reddy et al., 2018Reddy 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. http://dx.doi.org/10.1016/j.cryobiol.2018.08.005. PMid:30098997.
http://dx.doi.org/10.1016/j.cryobiol.201... ). In bovine, the HSP70 protein is widely distributed in spermatogonia, spermatids, and spermatozoa, and its localization can change during spermatogenesis, after ejaculation, as a result of capacitation and acrosome reactions, and during cryopreservation (Kamaruddin et al., 2004Kamaruddin M, Kroetsch T, Basrur PK, Hansen PJ, King WA. Immunolocalization of heat shock protein 70 in bovine spermatozoa. Andrologia. 2004;36(5):327-34. http://dx.doi.org/10.1111/j.1439-0272.2004.00629.x. PMid:15458553.
http://dx.doi.org/10.1111/j.1439-0272.20... ). HSP70 plays a role in the activation process of antioxidants, glutathione, taurine, creatine, zinc, vitamin E, vitamin C, and vitamin A and modulates the activation of heat shock factor and the subsequent synthesis of HSP70 (Singh et al., 2019Singh A, Kukreti R, Saso L, Kukreti S. Oxidative stress: a key modulator in neurodegenerative diseases. Molecules. 2019;24(8):1583. http://dx.doi.org/10.3390/molecules24081583. PMid:31013638.
http://dx.doi.org/10.3390/molecules24081... ; Kurashova et al., 2019Kurashova NA, Madaeva IM, Kolesnikova LI. Expression of heat shock proteins HSP70 under oxidative stress. Adv Gerontol. 2019;32(4):502-8. PMid:31800176.). In addition, the HSP70 protein is considered an essential element for the process of meiosis and maturation of sperm, as well as preventing apoptosis because it can activate the natural immune system and play a role in immunomodulation (Wang et al., 2020Wang 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. http://dx.doi.org/10.1016/j.gene.2020.145029. PMid:32758578.
http://dx.doi.org/10.1016/j.gene.2020.14... ). HSP70 also plays a role in sperm-egg membrane interactions (Spinaci et al., 2006Spinaci 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. http://dx.doi.org/10.2164/jandrol.106.001008. PMid:16870948.
http://dx.doi.org/10.2164/jandrol.106.00... ). Furthermore, HSP70 can increase membrane fluidity as a sperm protector in the female reproductive tract (Moein-Vaziri et al., 2014Moein-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. http://dx.doi.org/10.1530/REP-13-0631. PMid:24501193.
http://dx.doi.org/10.1530/REP-13-0631... ).

Overall, the HSP70 gene and protein could be detected in the sperm of the Bali bulls used in this study. Although not significantly different (P<0.05), HSP70 gene expression in bulls aged more than 12 years tended to be lower than in bulls aged less than nine years. It indicates the possibility of a decrease in HSP70 gene expression with increasing age in bulls. Gunes et al. (2016)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. http://dx.doi.org/10.1007/s10815-016-0663-y. PMid:26867640.
http://dx.doi.org/10.1007/s10815-016-066... reported that increasing age would impact oxidative stress, which causes an increase in lipid peroxidation and the formation of ROS in mitochondria. An increase in oxidative stress, which increases ROS, is reported to be associated with a decrease in HSP70 (2019). Although the expression of the HSP70 gene did not vary among ages, the research reveals a strong bond between HSP70 gene expression and field fertility. HSP70 gene expression has a positive correlation (P<0.01) with field fertility, which is also proven by the linearity test using the Scatter Plot curve, where there is a positive and linear relationship between these two parameters (Figure 5). It indicates that it could be a biomarker determining the fertility of bulls. The same finding was also reported by Rajoriya et al. (2014)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. http://dx.doi.org/10.1016/S2305-0500(14)60025-7.
http://dx.doi.org/10.1016/S2305-0500(14)... and Abdulla et al. (2022)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. http://dx.doi.org/10.14715/cmb/2022.68.4.14. PMid:35988278.
http://dx.doi.org/10.14715/cmb/2022.68.4... , who proposed that HSP70 is related to fertility rates. Bashiri et al. (2021)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. http://dx.doi.org/10.1007/s43032-020-00334-z. PMid:33140326.
http://dx.doi.org/10.1007/s43032-020-003... discovered that heat shock protein expression in sperm contributes to embryogenesis and performs a crucial function in fertilization, particularly during sperm-egg membrane interactions. Gene expression patterns or protein content changes can cause conception failure and reduce fertility.

There was no substantial difference (P>0.05) in the abundant HSP70 protein between the ages. In addition, the HSP70 protein does not have a close relationship with the fertility field and shows a negative linear curve (Figure 5). According to Vihinen (2015)Vihinen M. Types and effects of protein variations. Hum Genet. 2015;134(4):405-21. http://dx.doi.org/10.1007/s00439-015-1529-6. PMid:25616435.
http://dx.doi.org/10.1007/s00439-015-152... , initial genetic changes at the DNA and RNA levels can influence the final protein generated, affecting the number of protein molecules. Variations in these proteins could have a wide range of consequences on attributes such as sequence, conformation, stabilization, interaction, activity, concentration, and others (Vihinen 2015Vihinen M. Types and effects of protein variations. Hum Genet. 2015;134(4):405-21. http://dx.doi.org/10.1007/s00439-015-1529-6. PMid:25616435.
http://dx.doi.org/10.1007/s00439-015-152... ). Clancy and Brown (2008)Clancy S, Brown W. Translation: DNA to mRNA to protein. Nature Education. 2008;1(1):101. and Indriastuti et al. (2022)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. http://dx.doi.org/10.3390/ani12212955. PMid:36359078.
http://dx.doi.org/10.3390/ani12212955... also revealed that, in simple terms, protein is the result of translating genetic information from DNA transferred by mRNA through the transcription process. mRNA is subject to a wide range of degradative processes interconnected in translation events to regulate protein expression (Silver and Noble 2012Silver JT, Noble EG. Regulation of survival gene hsp70. Cell Stress Chaperones. 2012;17(1):1-9. http://dx.doi.org/10.1007/s12192-011-0290-6. PMid:21874533.
http://dx.doi.org/10.1007/s12192-011-029... ). Not all areas of the mRNA can be translated, particularly the untranslated region close to the 5' end of the molecule (UTR). Differences in the length of the 5' UTR can affect protein binding sites, affecting mRNA stabilization or translation utilization (Clancy and Brown 2008Clancy S, Brown W. Translation: DNA to mRNA to protein. Nature Education. 2008;1(1):101.). Translation of mRNA into protein is influenced by various factors, such as post-transcriptional gene silencing (PTGS), mRNA stability, and factors affecting mRNA stability, so a high mRNA level indicates a poor protein level if there are problems in the translation process (Egelkrout et al., 2012Egelkrout E, Rajan V, Howard JA. Overproduction of recombinant proteins in plants. Plant Sci. 2012;184:83-101. http://dx.doi.org/10.1016/j.plantsci.2011.12.005. PMid:22284713.
http://dx.doi.org/10.1016/j.plantsci.201... ).

Correlation test results between the HSP70 gene or protein and semen quality parameters did not show a strong relationship. It is different from previous studies, which found that there is a close relationship between HSP70 and motility (Pardede et al., 2023Pardede 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. http://dx.doi.org/10.3389/fvets.2023.1167594. PMid:37621869.
http://dx.doi.org/10.3389/fvets.2023.116... ; Zhang et al., 2015aZhang 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. http://dx.doi.org/10.17221/8239-CJAS.
http://dx.doi.org/10.17221/8239-CJAS... ; Hung et al., 1998Hung 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. http://dx.doi.org/10.1002/(SICI)1097-4644(19981001)71:1<21::AID-JCB3>3.0.CO;2-3. PMid:9736451.
http://dx.doi.org/10.1002/(SICI)1097-464... ), sperm morphological defects (Huang et al., 2000Huang 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. http://dx.doi.org/10.1016/S0378-4320(00)00175-5. PMid:10989233.
http://dx.doi.org/10.1016/S0378-4320(00)... ), acrosome integrity (Kamaruddin et al., 2004Kamaruddin M, Kroetsch T, Basrur PK, Hansen PJ, King WA. Immunolocalization of heat shock protein 70 in bovine spermatozoa. Andrologia. 2004;36(5):327-34. http://dx.doi.org/10.1111/j.1439-0272.2004.00629.x. PMid:15458553.
http://dx.doi.org/10.1111/j.1439-0272.20... ), and DNA fragmentation (Rajoriya et al., 2014Rajoriya 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. http://dx.doi.org/10.1016/S2305-0500(14)60025-7.
http://dx.doi.org/10.1016/S2305-0500(14)... ). Varghese et al. (2016)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. http://dx.doi.org/10.1016/j.theriogenology.2015.10.029. PMid:26607876.
http://dx.doi.org/10.1016/j.theriogenolo... also reported that there was a significant (P<0.05) decrease in HSP70 levels before freezing and after thawing. The difference in HSP70 levels was very substantial from the apical area on the head of cryopreserved sperm and compared to the same site of ​​sperm acrosome before the cryopreservation process. Furthermore, Grzmil et al. (2008)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. http://dx.doi.org/10.1095/biolreprod.107.065706. PMid:18256328.
http://dx.doi.org/10.1095/biolreprod.107... reported that the normal function of spermatozoa from each parameter of spermatozoa quality is managed by much more than one gene or protein and is a complicated task. Disruptions in every gene or protein that regulate the quality attributes of these sperms are very likely to impact decreasing bull fertility, which varies.

Conclusion

This study found a possible effect of age on a decrease in reproductive capacity, as seen from several parameters of semen quality, which decreased in Bali bulls aged more than 12 years. Bali bull age did not affect HSP70 gene expression and protein abundance. The HSP70 gene molecule has the prospect of being a valuable marker for determining Bali bull fertility.

Acknowledgements

The authors would like to sincerely thank the UPTD BIBDPTHPT Bali for providing bull data and the National Research and Innovation Agency (BRIN) for facilitating the implementation of this research.

References

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