Abstract

   Proteins assist sperm mature, transit the female reproductive tract,
   and recognise sperm oocytes. Indigenous Indonesian bulls, Madura bulls,
   have not been studied for reproductive proteomics. As local Indonesian
   beef livestock, Madura cattle assist in achieving food security; hence,
   their number must be improved. Thus, the identification of molecular
   proteomics-based bull fertility biomarkers is needed. This study aimed
   to characterise the sperm fertility function of the superior Madura
   bull (Bos indicus × Bos Javanicus) spermatozoa proteome. Frozen semen
   from eight Madura superior bulls (Bos indicus × Bos javanicus) aged 4–8
   years was obtained from the artificial insemination centre (AIC) in
   Singosari and Lembang. Madura superior bulls are those that have passed
   the bull breeding soundness evaluation. Frozen sperm were thawed and
   centrifuged at 3000 × g for 30 min. Proteins in sperm were
   characterised through proteomic analysis using liquid
   chromatography–tandem mass spectrometry (LC–MS/MS). The resulting gene
   symbols for each protein were then subjected to bioinformatics tools,
   including UniProt, DAVID, and STRING databases. Regarding sperm
   fertility, the analysis revealed that 15 proteins were identified in
   the sperm of Madura bulls. Amongst the identified proteins, the
   superior Madura bull sperm contained several motilities, energy-related
   proteins, and chaperone proteins. A substantial portion of
   characterised proteins are linked to metabolic pathways and the
   tricarboxylic acid (TCA) cycle, contributing to sperm energy
   production. In conclusion, the first in-depth proteome identification
   of sperm related to sperm quality and bull fertility of a unique
   indigenous Madura breed of Indonesia was performed using the LC–MS/MS
   proteomic method. These findings may serve as a reference point for
   further studies related to the functions of bovine sperm and biomarkers
   of fertility and sperm quality.

   Keywords: fertility, food security, LC–MS/MS, Madura bulls, sperm
   proteins fertility, proteomic, sperm proteins

1. Introduction

   Recent developments in proteomics have significantly impacted our
   understanding of how sperm become fertile ([41]1). One of the most
   differentiated cells is the sperm head, which has a highly compacted
   chromatin structure and an enormous midpiece that contains the
   machinery required to interact and transfer paternal genetic and
   epigenetic information to the oocyte ([42]2). Due to their high level
   of differentiation, sperm is suitable for studying proteomic sections,
   such as the sperm membrane, which is the most crucial part because of
   its role in interacting with the environment and the oocyte ([43]3).
   The fusion of a sperm and an oocyte requires complex membrane
   modifications of the sperm ([44]4). Spermatozoa proteins that regulate
   normal/abnormal sperm function have been identified through proteomic
   investigation ([45]5).

   Thus, bull fertility is linked to many proteins involved in
   spermatogenesis. The significance of specific proteins in controlling
   sperm quality and fertilization is unknown, although their availability
   and quantity may alter sperm fertility ([46]6). According to Peddinti
   et al. ([47]1), many bovine fertility protein markers have been
   reported. Major semen proteins, such as binding sperm protein (BSP) in
   Frisian Holstein cattle ([48]6), zona pellucida binding protein (ZPBP)
   in Bali Polled bulls ([49]7), A-kinase anchoring protein 3 in buffalo
   ([50]8), and osteopontin in Limousin cattle ([51]9), have been widely
   reported. The sperm proteome profile of mammals has been studied,
   including pigs ([52]10), equines ([53]11), sheep ([54]12), and bovines
   ([55]1). Nevertheless, the sperm proteome remains unknown in Madura
   bulls.

   Madura cattle (Bos indicus × Bos javanicus) are native to Madura,
   Indonesia. Small-scale producers raise Madura cattle as working cattle
   to maintain the regional culture, which still values Sonok and Karapan
   cattle ([56]13). In addition, Madura cattle are currently being
   developed as beef cattle using artificial insemination and reproduction
   technology. It also ensures food security for the Indonesian
   population. In particular, these cattle are highly valued for their
   unique reproductive traits, with regular calving even under low-input
   regimes and in dry and arid regions ([57]14). However, it remains
   unknown whether the proteome level of Madura bull sperm affects sperm
   function and fertility. Proteomics offers various methods. However,
   complex protein analysis using LC–MS has become the preferred
   analytical technique for quantitative proteomics ([58]15, [59]16).

   Ultimately, acquiring knowledge about the proteome of spermatozoa can
   provide a holistic understanding of reproductive processes, not only
   specific to the Madura bull breed but also applicable to bovine
   species. This study aims to provide a comprehensive profiling of the
   sperm proteins associated with fertility function in the superior
   Madura bull (Bos indicus × Bos javanicus) spermatozoa proteome.
   Additionally, we used the literature to explain how Madura bull sperm
   proteins affect bull fertility. Further, proteomic approaches have made
   it possible to identify proteins that have the potential to function as
   indicators of male fertility, as well as proteins that are involved in
   the functional characteristics of sperm.

2. Materials and methods

2.1. Frozen semen samples

   A total of 8 superior Madura bulls (Bos indicus × Bos javanicus) aged 4
   to 8 years with spermatozoa motility >70%, based on secondary data from
   each AIC in Lembang and Singosari, Indonesia, were included in this
   study. Frozen semen from each of the eight Madura superior bulls was
   obtained from the National AIC in Singosari and Lembang Bank of Semen.
   Madura superior bulls are those that have passed the bull breeding
   soundness evaluation. The Animal Care and Use Committee excluded this
   study from its ethical review because artificial vaginal semen
   collection did not alter the physiology of animals. The study was
   directed by veterinarians from both institutions and followed SNI ISO
   9001: 2015 No. 824 100 16072 at Lembang AIC and SNI ISO 9001: 2015 No.
   G.01-ID0139-VIII-2019 at Singosari AIC. The ethics committees of
   Lembang AIC and Singosari AIC provided ethical guidance and sanctions
   for the responsible collection of bull sperm. Additionally, an
   experienced bull technician collected sperm using an artificial vagina.

   Semen samples from Lembang AIC were processed according to the AIC’s
   standard operating procedure (SOP), which used skim milk as the
   extender. The formulation of the skimmed milk diluent in a volume of
   1000cc consisted of 100g of skimmed milk, 960cc of distilled water, and
   an antibiotic solution containing 3000000 IU of penicillin, 3g of
   streptomycin, and 30cc of distilled water. The ratio of skimmed milk
   diluent to antibiotic was 100:1. In contrast, the semen samples
   obtained from Singosari were cryopreserved utilising tris egg yolk as a
   cryoprotectant. The tris egg yolk extender was formulated using the
   following components: 20% egg yolk, 1.6% tris aminomethane, 1.4%
   lactose, 2.5% raffinose, 0.9% citric acid, and an antibiotic mixture
   consisting of 1000000 IU/L of penicillin, 1g/L of streptomycin, and
   1g/L of distilled water. The AIC in Lembang and Singosari employ
   extenders consisting of skim milk and tris egg yolk supplemented with
   antibiotics. The extender utilised in every artificial insemination
   facility serves as a cryoprotectant, safeguarding sperm cells during
   the freezing process.

   Additionally, it enhances semen volume, sustains sperm viability, and
   regulates sperm pH. Consequently, the inclusion of an extender is
   imperative in the manufacture of frozen semen to ensure the
   preservation of sperm quality. The cryopreserved sperm samples were
   transferred with a specialised transport container that maintained a
   temperature of −196°C through liquid nitrogen. The pieces were placed
   within the container for subsequent examination.

2.2. Sperm protein isolation

   The collected semen samples of about ive straws of frozen semen from
   each of the eight bulls were washed in 2 mL of phosphate buffer saline
   and centrifuged twice (3000 × g for 10 min, 4°C) to clean and separate
   the spermatozoa, seminal plasma, and extender of frozen semen. The
   resulting sperm cell pellet was then resuspended in cell lysis buffer
   (2% SDS in 62.5 mM Tris–HCl, pH 6.8, 1.0 mM phenylmethanesulfonyl
   fluoride, and 23 mM benzidine as a protease inhibitor) and stored at
   −20°C for protein extraction. The sperm pellet was vortexed for 10 min
   before protein extraction. Next, sonication was used thrice for 20 s
   each to dissolve the protein. After centrifuging the cell lysates
   (10,000 × g, 10 min), protein lysates were isolated. The Bradford
   method ([60]17) was used to estimate the total protein yield of the
   sperm protein lysate. The Bradford protocol was performed using the
   Coomassie protein kit instructions (Merck, Darmstadt, Germany). The
   data were processed using ThermoScan RE software version 3.2 Multiskan
   Go (Thermo Fisher Scientific, Waltham, Massachusetts, United States).
   Pools of sperm protein containing equal amounts from three samples were
   frozen at −20°C for later use. A summary of the experimental design is
   shown in [61]Figure 1.

Figure 1.

   [62]Figure 1
   [63]Open in a new tab

   Schematic workflow for the proteomic analysis of Madura bull
   spermatozoa. (A) Sperm protein extraction. (B) Sperm protein
   identification.

2.3. Fractionation by one-dimensional sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (1D-SDS–PAGE) and in-gel digestion

   Following the separation of 35 μg of sperm protein (an equivalent
   quantity of protein amalgamated from three cases) by 12.5% sodium
   dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE), the gel
   was stained with colloidal Coomassie brilliant blue R-250 (CAS number:
   6104-59-2) from HiMedia. Subsequently, the gel bands were excised and
   digested using a previously described protocol ([64]18). The proteins
   were reduced using 0.5 M dithiothreitol (DTT) at 55°C for 25 min,
   followed by alkylation with 14 mM iodoacetamide (IAA) for 40 min at
   room temperature without light. Gel fragments were treated with trypsin
   (Promega, Madison, Fitchburg, WI, USA) at a ratio of 1:50 (trypsin:
   protein) at 4°C for 30 min. The gel fragments were incubated for 18 h
   at 37°C in a ThermoMixer shaker incubator block (Eppendorf). The
   reaction in each vial was terminated by adding 1% trifluoroacetic acid
   (TFA). An extraction buffer (2% TFA in 20% acetonitrile) was used to
   extract peptide digests from the gel pieces, followed by 70%
   acetonitrile. The obtained peptide digests were subjected to a drying
   process and subsequently purified using a C18 spin column for
   desalination (Thermo Scientific, Pierce Biotechnology, N Meridian Rd.,
   Rockford, IL, USA) and then preserved at −20°C until LC–MS/MS analysis.

2.4. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis

   The peptides obtained from each sample were subjected to LC–MS/MS
   analysis using an Ultimate 3000 Nano LC system coupled with a
   Q-Exactive Plus Orbitrap HRMS system (Thermo Fisher Scientific, Bremen,
   Germany). The peptides were introduced onto a preanalytical column with
   dimensions of 75 μm ID, 15 cm length, and 100 pore size, packed with
   Acclaim PepMap C18 2 μm particles. The solvent used for loading was
   solvent A, consisting of 0.1% formic acid, and the flow rate was set at
   300 nL/min. A Q-Exactive Plus Orbitrap mass analyser was used for
   subsequent analysis. The peptides were subsequently resolved on an
   analytical column (50 cm 75 m ID, Pep Map RSLC C18 2 m) at a flow rate
   of 300 nL/min for 120 min using an increasing gradient of 5–35% solvent
   B (98% acetonitrile and 0.1% formic acid). Peptide signals were
   collected using an LTQ-Orbitrap mass spectrometer (Thermo Fisher
   Scientific, Bremen, Germany). The MS spectra within the 200–2000 m/z
   range were obtained using an Orbitrap analyser with a resolution of
   30,000 (at m/z 400). Subsequently, ten precursor MS scans were
   conducted using collision-induced dissociation fragmentation at 35%
   normalised collision energy ([65]19).

2.5. Protein database searching and bioinformatics analysis

   Protein analysis was performed on proteins that contained at least one
   unique peptide. The exclusion of specific peptides based on the
   “minimum two-peptide rule may result in the perpetual disregard of
   genuine peptides, and proteins must have a sequence score HT > 0. Thus,
   we manually confirmed single peptide spectral match (PSM)-identified
   protein MS/MS spectra. Protein data were acquired and analysed using
   the UniProt bovine protein database ([66]http://www.uniprot.org) and
   Proteome Discover version 2.2 software (Thermo Fisher Scientific).
   Furthermore, the discovered proteins from spermatozoa were also put
   into functional groups based on Gene Ontology (GO) using the DAVID web
   resource ([67]https://david.ncifcrf.gov/tools.jsp). The interactions
   between the proteins were obtained using STRING version 11.0
   ([68]https://string-db.org/).

3. Result

3.1. Protein characterisation of Madura bull spermatozoa

   LC–MS/MS identified Madura bull sperm protein markers of fertility and
   environmental compatibility. All the databases contained in the
   database were only Bos taurus origin proteins since Bos indicus × Bos
   javanicus proteins are limited. This study analysis identified 15
   proteins in Madura bull (Bos indicus × Bos Javanicus) sperm using
   UniProt and DAVID software ([69]Table 1). Fifteen proteins were
   identified that were associated with sperm function categories
   ([70]Table 1), including adenosine triphosphate (ATP) synthesis
   activity, cellular metabolic processes, cilia/flagella, sperm motility,
   capacitation and acrosome reaction, sperm-egg fusion, spermatogenesis
   and fertilization, and chaperone proteins. Functional annotation of
   proteins from Madura bull spermatozoa was further carried out using
   DAVID software and divided into several categories: “Biological
   Process” (BP), “Cellular Component” (CC), and “Molecular Function” (MF)
   ([71]Figure 2). Although different categories were created for each
   division, the most numerous were those involved in the generation of
   precursor metabolites and energy (16%), urine ribonucleotide metabolic
   processes (16%), ATP metabolic processes (13%), and pyruvate metabolic
   processes (10%) in the case of BP ([72]Figure 2A); cytoplasm (24%),
   supramolecular fibre (21%), and cell surface and microtubule (10%) in
   CC ([73]Figure 2B); and ATP-binding proteins (16%), ATPase activity
   (12%) and unfolded protein binding (8%) in MF ([74]Figure 2C).

Table 1.

   Proteins identified in Madura bull sperm associated with sperm
   function.
   Protein accession Gene symbol Protein name Function Molecular weight
   (kDa)
   A0A4 W2HIH5 AKAP4 A-kinase anchoring protein 4 flagellated sperm
   motility, motile cilium assembly 99.6
   A0A4 W2BT96 ATP5F1A ATP synthase F1 subunit alpha lipid metabolic
   process, ATP synthase activity 63.1
   A0A452DII8 ATP5F1B ATP synthase F1 subunit beta ATP synthesis activity,
   Hydrogen ion transport, Ion transport 62.2
   A0A6P5BW43 ACO2 aconitase 2 tricarboxylic acid cycle, citrate metabolic
   process 85.3
   A0A4 W2CUL5 COL4A2 collagen type IV alpha 2 chain cell membrane
   receptors, metabolic processes, spermatozoa differentiation, 179.5
   A0A4 W2EVB5 C1orf56 Chromosome 1 open reading frame 56 Capacitated
   sperm and acrosome reaction 37.9
   A0A3Q1M0V5 ENO3 Enolase 3 glycolytic process, pyruvate metabolism, ATP
   formation from ADP 62.1
   A0A4 W2CEF3 GAPDHS glyceraldehyde-3-phosphate dehydrogenase,
   spermatogenic glucose metabolism, glycolytic process, formation of ATP
   from ADP, spermatozoa energy precursor 44.1
   A0A4 W2D4U4 HSPA2 heat shock protein family A (Hsp70) member 2 male
   meiosis I, spermatogenesis, spermatid development, response to heat and
   cold stress, positive regulation of G2/M transition of the mitotic cell
   cycle, cell differentiation, positive control over ATPase activity,
   protein folding mediated by chaperones that require cofactors, the
   reaction of cells to unfolded proteins, and protein refolding 69.81
   A0A4 W2DHF8 HSPA9 heat shock protein family A (Hsp70) member 9 ATP
   binding, metabolic processes, mitochondrial protein folding, enzyme
   binding, stress response 73.7
   A0A4 W2I3C7 SPAM1 sperm adhesion molecule 1 (Hyaluronidase) Sperm egg
   recognition, fertilization 62.4
   A0A6P5CLI1 ODF2 the outer dense fiber of sperm tails 2 spermatogenesis,
   cell differentiation, cilium organisation, regulation of cilium
   assembly, 105
   A0A6P5BWX7 PDHA2 pyruvate dehydrogenase E1 subunit alpha 2 Carbohydrate
   metabolism, Glucose metabolism, 43.3
   [75]Q3MHM5 TUBB4B tubulin beta 4B class IVb microtubule-based process,
   Cytoskeleton 49.8
   F6RP72 TUBB1A Tubulin alpha chain microtubule-based process,
   Cytoskeleton 50.9
   [76]Open in a new tab

Figure 2.

   [77]Figure 2
   [78]Open in a new tab

   Bar chart representing the gene ontology annotations of proteins
   identified in Madura bull spermatozoa according to biological processes
   (A), cellular components (B), and molecular functions (C).

3.2. Protein interaction networks and pathway enrichment analysis

   All proteins identified in Madura bull spermatozoa were then searched
   using STRING software (version 11.0) for protein–protein interaction
   network analysis. Light blue lines show protein nodes, whereas pink
   lines reflect experimentally established interactions. The expected
   interactions are green for the gene neighbourhood, red for gene fusion,
   and dark blue for gene co-occurrence. Other protein relationships are
   shown by light green text mining, black co-expression, and blue protein
   homologies. A coloured node shows the query protein, and a white node
   leads the second shell of the interaction. Proteins with unknown 3D
   conformations had empty nodes, whereas those with known conformations
   had full nodes. The interactions between the 15 identified proteins are
   shown in [79]Figure 3. Fifteen proteins were directly or indirectly
   connected through one or more interacting proteins, indicating
   functional links. We categorised proteins using the Kyoto Encyclopaedia
   of Genes and Genomes (KEGG) pathway terminology to study Madura bull
   sperm pathways. As expected, many of the discovered proteins were
   involved in metabolic pathways. The discovered proteins were associated
   with the TCA cycle ([80]Figure 4). The sperm head, mitochondria
   (mid-piece), and flagellum/tail expressed the majority of these
   proteins ([81]Figure 5).

Figure 3.

   [82]Figure 3
   [83]Open in a new tab

   STRING protein–protein interaction network showing the interactions of
   the spermatozoa proteins identified in Madura bull sperm.

Figure 4.

   [84]Figure 4
   [85]Open in a new tab

   Pathway analysis using the KEGG pathway database identified proteins
   involved in the TCA cycle. The red star denotes the detected proteins.

Figure 5.

   [86]Figure 5
   [87]Open in a new tab

   Distribution of sperm protein of Madura bull (Bos indicus × Bos
   javanicus).

4. Discussion

   Fifteen proteins found in the sperm of Madura bulls were found to be
   related to fertility based on LC–MS/MS analysis ([88]Table 1). The
   utilisation of known molecular weight standards for calibration is a
   fundamental aspect of size-exclusion chromatography that provides
   insights into the distribution of molecular weights. The current study
   has demonstrated that Madura bull sperm exhibits a molecular weight
   distribution of proteins within the 40 to 180 kDa range. This study
   utilised GO annotation to ascertain the cellular localisation,
   biological processes, and molecular functions of proteins specific to
   the sperm of Madura bulls.

4.1. Protein related to sperm fertility function

   Effect of sperm motility on semen quality Male infertility is often
   attributed to reduced sperm motility. Therefore, the motility proteins
   of the Madura bull breed must be identified. We found many of these
   proteins in the spermatozoa. SPAM1 is a transmembrane protein present
   in ejaculated bull spermatozoa. It originates from the testis and
   occupies the anterior head region, where the orientation of the
   C-terminus with the zonal-binding domain facilitates interaction with
   zona pellucida after the acrosome reaction ([89]20). This protein has
   also been detected in male sperm ([90]21). The roteomee analysis
   carried out in this study from the sperm of Madura Bull also revealed
   the existence of the hyaluronidase protein, also known as SPAM1,
   isoform 62.4 kDa.

   The flagellum of mammalian spermatozoa has intricate supplemental
   structures around the core axoneme, including the outer dense fibers
   (ODFs). ODFs protect the sperm tail against shear stress that may arise
   during epididymal transit and ejaculation. Four major proteins (ODF1,
   ODF2, ODF3, and ODF4) are amongst more than 14 polypeptides produced by
   mammalian ODFs. In mice, disrupting ODF2 expression lowers sperm
   motility, which is consistent with the characteristics of
   asthenozoospermia ([91]22). Moreover, the cervical midpiece junction
   requires the presence of ODF2, which comprises part of the centrosome
   and is separated from the flagellum ([92]23). Our analysis identified
   ODF2 in Madura bull spermatozoa.

   In addition, we found that enolase protein, previously discovered in
   the plasma membrane of sperm from Bali bulls ([93]24), was present in
   sperm samples from Madura bulls. During glycolysis and gluconeogenesis,
   enolase reversibly converts 2-phosphoglycerate into
   phosphoenolpyruvate. This enzyme produces sperm motility energy
   ([94]25). Xi et al. ([95]26) found a significant positive correlation
   between ENO3 and sperm motility parameters in sturgeons. Similarly, He
   et al. ([96]25) discovered ENO3 in the midpiece and tail of ram sperm,
   which strongly correlated with sperm motility. The presence of ENO3
   expression in Madura cattle sperm and its correlation with sperm
   function, particularly motility, can be readily explained.

   The present study revealed the detectability of AKAP 4 in Madura bull
   sperm using LC–MS/MS. AKAP4, a marker involved in flagellar structure
   and motion, is associated with the equine sperm tail ([97]27) and is
   substantially preserved in mice ([98]28), bulls ([99]29), and humans
   ([100]30). AKAP4 attaches cAMP-dependent protein kinase A (PKA) to
   different subcellular locations. In the sperm fibrous sheath, AKAP4
   binds PKA and interacts with other proteins to regulate motility.
   AKAP4, a critical fibrous sheath protein, bundles glycolytic enzymes
   and phosphorylation-signalling cascade components to supply a localised
   source of ATP and govern flagellar motion, sperm motility, and
   hypermotility. Many glycolytic enzymes closely linked to the fibrous
   sheath at the primary component of the flagellum are required for sperm
   motility ([101]27).

   Tubulins were also found to be another type of protein that contributes
   to sperm motility that was uncovered in this study. Tubulins are
   proteins found in the microtubules of sperm ([102]30). Two tubulin
   proteins were identified as present in the sperm of the studied Madura
   bull samples. These proteins are known as TUBA1A and TUBB4B. These
   proteins are necessary for cilia and flagella formation; hence, their
   importance cannot be overstated.

4.2. Chaperone proteins: heat shock proteins

   Heat stress affects cattle productivity and reproduction. Heat stress
   damages developing spermatozoa. However, heat tolerance can be
   inherited by offspring ([103]3). According to the relevant literature,
   Madura cattle are a native breed of beef cow thought to have developed
   on the dry and barren island of Madura ([104]3). Madura Island receives
   1600 mm of rain annually and is hot and arid ([105]31). HSPs are linked
   to heat tolerance and reproductive performance ([106]3, [107]32). In
   this study, HSPA9 and HSPA2 were found to be ubiquitously expressed.
   HSPA9, found in sperm mitochondria, significantly reduces the effects
   of heat stress in tropical cattle ([108]33, [109]34). It is also known
   to regulate sperm motility through metabolic processes, ATP binding,
   and folding of mitochondrial proteins ([110]35).

   HSPA2 (also known as HSP70-2) was discovered in this study, which is
   consistent with previous findings in Australian Brahman bulls (Bos
   indicus) ([111]36) and Zebu ([112]37). HSP70-2 (heat shock protein
   70–2) was found to be expressed in the sperm nucleus as well as in
   various other sperm organelles, including the mitochondria and
   flagellum ([113]Figure 5). HSP70-2 is a component of the chromatin
   structure and contributes to gene regulation, particularly in the
   folding and unfolding of proteins. This HSPA2 sperm protein may also be
   crucial in protecting the sperm of tropical acclimated bulls such as
   Madura from heat or environmental stress. HSP70 is also an excellent
   predictor of thermotolerance and thermoresistance ([114]38). The
   potential use of HSP70 as a biomarker for animal fertility and
   thermotolerance has been suggested ([115]39). Therefore, HSP70-2 is
   recognised as a dual-functioning gene ([116]3).

4.3. Pathway enrichment

   The enriched pathways were also analysed for the identified proteins in
   the Madura bull spermatozoa. The present study revealed that 16% of the
   proteins were involved in the biosynthesis of precursor metabolites and
   energy. Only metabolic pathways were included in the list of the
   significant ways enriched by the proteins. Energy metabolism is
   essential for sperm development. Sperm require ATP, which is most
   likely to maintain morphological changes during the spermatid stage,
   for the degradation and synthesis of active proteins ([117]40). ATP
   also serves as the molecular motor that provides the necessary energy
   for flagellar movement in all kinetics-related biochemical activities.
   The idea that mitochondria in the sperm midpiece drive mammalian sperm
   is evolving, as evidence suggests that glycolysis is the preferred
   metabolic pathway to maintain sperm motility in many species ([118]41).
   Thus, the current study’s results are in line with earlier research
   showing that ATP synthases are hub proteins found in the sperm of rams
   that are comparatively less fertile and may regulate how much ATP is
   used by sperm throughout the fertilization process ([119]42). Existing
   data indicate that ATP production in sperm is facilitated by both
   glycolysis and mitochondrial respiration. These processes are
   interdependent and regulate sperm function based on the availability of
   energy substrates in the surrounding environment. An alternative
   glycolysis pathway for ATP production is also found in stallion
   spermatozoa ([120]43). This study presents novel findings that suggest
   that Madura sperm are dependent on mitochondrial ATP synthesis,
   specifically regarding the roles of ATPF51A and ATP5B in sperm function
   and fertility.

   Further, glucose utilisation is restricted during spermatogenesis, and
   lactate and pyruvate are favoured as substrates for energy synthesis
   ([121]19). The generation of ATP in mammalian sperm is attributed to
   the energy metabolism pathway. This pathway exhibits subcellular
   compartmentalisation, with oxidative phosphorylation (OXPHOS)
   predominantly occurring in the sperm midpiece and glycolysis in the
   principal piece ([122]40). Glycolysis is a significant mechanism for
   facilitating the transport of ATP along the flagellum. Westhoff and
   Kamp ([123]44), as well as Welch et al. ([124]45), have reported the
   presence of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in
   significant quantities within the fibrous sheath of sperm from
   different mammalian species, including humans. GAPDH is a NAD-dependent
   glycolytic enzyme responsible for facilitating the conversion of
   glyceraldehyde 3-phosphate (GAP) to 1,3-biphosphoglycerate (1,3 BPG).
   Notably, one of the isoforms of GAPDH, known as GAPDHS, has been
   identified in this study.

   Moreover, Zini et al. ([125]46) found that an augmentation in the
   activity of pyruvate dehydrogenase complexes can increase
   pyruvate-lactate usage, thus producing a more significant amount of
   NADH. NADH oxidase can utilise the NADH produced to generate reactive
   oxygen species (ROS) necessary for the capacitation process. NADH
   oxidase activity and ROS formation have also been associated with the
   motility of human spermatozoa. In this way, GAPDHS contributes
   significantly to energy metabolism. Elkina et al. ([126]47) found
   GAPDHS in the testes of humans and rats; the enzyme is primarily
   present in the cytoplasm of all spermatogenic cells, whereas it is
   localised in the sperm tail in the epididymis. This finding provides
   support for this theory. This previous study by Kumar et al. ([127]48)
   also established the involvement of extramitochondrial localised
   pyruvate dehydrogenase complex (PDHA) in the process of sperm
   capacitation, as well as the significance of pyruvate in the overall
   energy metabolism of mammalian sperm. The metabolic pathway encompassed
   the GAPDHS and PDHA2 proteins, both detected in the sperm of Madura
   bulls in the present study.

   In agreement, energy metabolism plays a critical role in facilitating
   sperm function. The TCA cycle is the primary energy source for
   spermatids, although glycolytic and pentose phosphate pathways also
   play a role in energy synthesis in spermatozoa. The TCA cycle generates
   adenine, which is converted to ATP by the electron transport chain of
   the OXPHOS pathway. These proteins may be involved in creating the
   acrosome and activities there, both of which require energy provided by
   oxidative phosphorylation. ATP is then delivered to the microtubules
   responsible for supporting various sperm functions ([128]49). In this
   study, two candidate proteins related to the tricarboxylic acid cycle
   pathway were PDHA2 and ACO2.

   It was intriguing to discover that specific proteins are involved in so
   many energy metabolism processes, suggesting that a single protein may
   not perform glycolysis or OXPHOS as the primary actor in maintaining
   sperm functionality. The current study shows that proteomic methods may
   provide a reliable source for sperm protein detection and improve gene
   ontology comprehension. Therefore, we used high-throughput LC–MS/MS to
   develop a proteomic profile of Madura bull sperm connected to
   reproductive characteristics. Preserving superior native germplasm and
   preventing gene pool depletion requires studying protein differences in
   different breeds. Thus, investigating Madura bull protein composition
   can help us comprehend native Indonesian bull sperm biology. Finally,
   knowing the proteome of ejaculated spermatozoa can help us understand
   the reproductive process in Madura bull and bovine in general.

5. Conclusion

   We examined the sperm proteomes of Madura cows (Bos indicus × Bos
   javanicus). The use of several methods to prepare samples has made it
   easier to identify proteins in sperm. Most of the proteins identified
   in this study are essential for bull breeding. We believe that
   information about the sperm proteome of the superior Madura bull, a
   breed with good reproductive traits, will speed up future studies on
   bull fertility. Additionally, this would enable the creation of
   molecular tools for accurately selecting bulls to conserve indigenous
   Indonesian cattle breeds and bovines.

Data availability statement

   The original contributions presented in the study are included in the
   article/supplementary material, further inquiries can be directed to
   the corresponding authors.

Ethics statement

   Ethical approval was not required for the studies on animals in
   accordance with the local legislation and institutional requirements
   because only commercially available established cell lines were used.

Author contributions

   ZR: Conceptualization, Data curation, Writing – original draft, Writing
   – review & editing, Formal analysis, Investigation, Methodology. BP:
   Data curation, Funding acquisition, Project administration, Validation,
   Writing – original draft, Writing – review & editing, Formal analysis,
   Investigation, Methodology, Visualization. EK: Writing – review &
   editing. MG: Writing – review & editing. TM: Writing – review &
   editing. LT: Writing – review & editing, Conceptualization,
   Supervision. DS: Conceptualization, Supervision, Writing – review &
   editing. MU: Conceptualization, Supervision, Writing – review &
   editing. BP: Conceptualization, Data curation, Funding acquisition,
   Project administration, Supervision, Validation, Writing – original
   draft, Writing – review & editing. SS: Methodology, Supervision, Formal
   analysis, Validation, Investigation, Funding acquisition, Writing –
   review & editing.

Acknowledgments