Lack of differential sperm acrosome-associated 1 gene expression between X- and Y-bearing spermatozoa in Bali bulls (Bos javanicus domesticus)

Abstract

This study aimed to determine whether the sperm acrosome-associated 1 (SPACA1) gene exhibits differential expression between X- and Y-bearing spermatozoa of Bali bulls and assess its potential as an RNA biomarker for sperm sexing. Sexed frozen semen (X or Y fractions) from Bali bulls was obtained from the Singosari National Artificial Insemination Center, Malang, Indonesia. Post-thaw semen quality was evaluated for motility, viability, abnormality, concentration, and plasma membrane integrity. Total RNA was extracted separately from X and Y fractions using easy-BLUE reagent, and SPACA1 expression was quantified by RT-qPCR using GAPDH as an internal control. Relative expression was calculated by the 2^−ΔΔCt method, and group differences were analyzed with the Mann-Whitney U test. The SPACA1 expression was numerically higher in X-bearing than Y-bearing spermatozoa (1.00 versus 0.51), but the difference was not significant. Thus, SPACA1 transcripts do not provide a reliable molecular marker for sperm sex differentiation. Future work should focus on protein-level characterization of SPACA1 and exploration of sex-biased surface proteins for immunological sexing. This research supports Sustainable Development Goal 2: Zero Hunger, by contributing to reproductive efficiency in livestock, and Sustainable Development Goal 12: Responsible Consumption and Production, through development of cost-effective, sustainable sperm sexing technologies.

Introduction

Sperm sexing is a valuable biotechnology in modern cattle reproduction management, enabling the preferential production of offspring of a desired sex to match breeding goals and economic incentives. In dairy cattle systems, the ability to produce female calves supports herd replace-ment and milk productivity, whereas in beef operations, male calves offer greater growth rates and economic returns. Currently, the most accurate method for sperm sexing involves flow cytometric sorting based on DNA content difference between X- and Y-chromosome-bearing spermatozoa. Although effective, this technique requires sophisticated equipment, is costly, and may negatively impact sperm quality due to the mechanical stress, fluorescence exposure, and cryopreservation processes.^1,2

To overcome these limitations, molecular approaches targeting intrinsic sex-specific markers in spermatozoa are being explored. One promising avenue involves the identification of sex-biased proteins located on the sperm surface, which could be used in immunological sorting via antibodies distinguishing X- from Y-bearing spermatozoa.^3,4 Compared to flow cytometry, antibody-based sorting may offer a less damaging, more scalable, and cost-effective strategy suitable for field implementation.

Studies on spermatozoal transcriptomes have identified putative sex-linked RNAs in bovine species.⁵ However, mature mammalian sperm cells are transcriptionally quiescent due to the extensive chromatin condensation during spermiogenesis, limiting their RNA content to residual transcripts from earlier spermatogenic stages.^6,7 This biology constrains the reliability of RNA-based sexing and highlights the importance of validating any potential markers at the protein level.

Sperm acrosome-associated 1 (SPACA1) is a gene encoding a transmembrane protein critical for acrosome formation and sperm-egg fusion.⁸ In mouse models, SPACA1 deficiency leads to acrosome malformation and subfertility, confirming its essential role in fertilization.⁹ In cattle, SPACA1 has been proposed as a marker of sperm integrity, with decreased expression linked to the subfertility in bulls. Specifically, a study in Bali cattle (Bos javanicus domesticus), an indigenous Indonesian breed of high conservation value, showed that reduced SPACA1 mRNA and protein levels were associated with poor semen quality and low fertility.¹⁰ These findings raise the possibility that SPACA1 may also exhibit sex-biased expression patterns in spermatozoa, thus supporting its potential use in sperm sexing applications.

To date, no study has assessed whether SPACA1 expression differs between X- and Y-bearing spermatozoa in Bali bulls. Investigating this expression difference at the mRNA level would provide insight into its suitability as a molecular sexing biomarker and guide future development of immunoselection techniques targeting SPACA1.

Accordingly, the present study aimed to evaluate SPACA1 transcript abundance in X- and Y-bearing spermatozoa of Bali bulls using RT-qPCR. The outcomes of this study will contribute to the understanding of SPACA1’s role in sex differentiation and its relevance for sperm selection strategies in local cattle breeds. This approach not only enhances reproductive efficiency but also aligns with global goals to improve food security and sustainable livestock management, as highlighted in Sustainable Development Goal 2: Zero Hunger and Sustainable Development Goal 12: Responsible Consumption and Production.

Materials and Methods

Semen source and sexing procedure.

Sexed frozen semen samples of Bali bulls (B. javanicus domesticus) were obtained from Singosari National Artificial Insemination Center, Malang, Indonesia. Two separate semen batches were used, derived from a single bull (Code: 11197), and sexed via separation by swim-up through discontinuous albumin gradient followed by cryopreservation into X-bearing (Batch: VV0705) and Y-bearing (Batch: XX0520) fractions. All procedures involving animal material were conducted in accordance with the Animal Ethics Committee of the Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia, and approved under protocol number of 1.KEH.90.06.2025.

Post-thaw semen quality assessment.

Semen straws were thawed at 37.00 ˚C for 30 sec and assessed for motility under light microscopy (CX23; Olympus, Tokyo, Japan). Viability was evaluated using Eosin-Nigrosin staining method, classifying live spermatozoa as unstained and dead spermatozoa as stained.^11-13 Morphological abnormalities were identified under light microscopy based on head, midpiece, and tail defects.^14,15 Sperm concentration was determined via hemocytometer following 1:100 dilution, using the formula: N × 5 × 100 × 10⁴ (N = mean sperm count per grid).¹⁶ Plasma membrane integrity was assessed using the hypo-osmotic swelling test with citrate-fructose solution (0.735 g sodium citrate and 1.351 g fructose in 100 mL distilled water), with intact membranes indicated by tail curling after 1 hr incubation at 37.00 ˚C.^17,18 All evaluations were performed in duplicate per sample.

RNA extraction and quality verification.

Total RNA was extracted from 3.00 -10.00 × 10⁶ spermatozoa per fraction using easy-BLUE Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam-si, South Korea), following the manufacturer’s protocol. Homogenates were stored at 4.00 ˚C and processed within one week. The RNA integrity was examined using 1.00% agarose gel electrophoresis; the presence of distinct 28S and 18S ribosomal RNA bands with an approximate 2:1 ratio was interpreted as intact RNA.^19,20

cDNA Synthesis and RT-qPCR.

Reverse transcription was performed using a first-strand cDNA synthesis kit (Thermo Fisher Scientific, Waltham, USA) with oligo(dT) primers (Thermo Fisher Scientific). Quantitative PCR (qPCR) was conducted using SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA) and primers specific for SPACA1 and GAPDH as the internal control. Primer specificity was validated by melt curve analysis. Relative expression levels were calculated using the 2^−ΔΔCt method.²¹ Each reaction was performed in triplicate as technical replicates for each sperm fraction, and experiments were repeated using three independent preparations per group as biological replicates (n = 3).

Statistical analysis.

The normality of relative expression data was evaluated using the Shapiro-Wilk test. Due to the non-normal distribution, differences in SPACA1 expression between X- and Y-bearing spermatozoa were analyzed using the non-parametric Mann-Whitney U test. A p-value < 0.05 was considered statistically significant. Data analysis was conducted using SPSS Software (version 25.0; IBM Corp., Armonk, USA).

Results

Post-thaw semen quality.

The sexed semen samples of Bali bulls met the minimum post-thaw quality standards for artificial insemination use. Both X- and Y-bearing sperm fractions demonstrated high motility and viability with minimal morphological abnormalities and acceptable plasma membrane integrity, indicating cryopreservation success. Mean values for motility, viability, abnormalities, plasma membrane integrity, and concentration are summarized in Figure 1. Representative fields from Eosin-Nigrosin staining (Fig. 2) showed a predominance of viable sperms (unstained), morphological assessment revealed low incidence of head-tail disjunctions, and hypo-osmotic swelling test test results further confirmed plasma membrane integrity through characteristic tail coiling in most cells.

Fig. 1.

Post-thaw semen quality parameters of Bali bull. Bar charts represent the mean ± standard deviation values of sperm motility (%), viability (%), morphological abnormalities (%), plasma membrane integrity (%), and concentration (×10⁶ sperm mL^-1) in sexed semen samples. All parameters met the minimum standards required for artificial insemination.

Fig. 2.

Post-thaw microscopic evaluation of Bali bull spermatozoa (400 ×). A) Eosin-Nigrosin staining showing viable spermatozoa (unstained; black arrows) and non-viable spermatozoa (stained; red arrows); B) Morphological abnormality illustrating head-tail disjunction (red arrow); C) Hypo-osmotic swelling test demonstrating a spermatozoon with a curled tail (black arrow), indicating intact plasma membrane; D) The HOST showing a spermatozoon with a straight tail (red arrow), indicating damaged plasma membrane (Scale bars = 10 µm).

RNA integrity verification.

Electrophoretic analysis of total RNA extracted from both sperm populations revealed sharp 28S and 18S rRNA bands with an approximate 2:1 intensity ratio, indicative of intact RNA suitable for RT-qPCR (Fig. 3). The absence of smearing or degradation across samples confirmed extraction quality.

Fig. 3.

Agarose gel electrophoresis of total RNA extracted from X- and Y-bearing spermatozoa; Lanes 1-2: X-bearing sperm RNA samples; Lanes 3-4: Y-bearing sperm RNA samples. Clear 28S and 18S rRNA bands indicate RNA integrity.

RT-qPCR assay performance.

The SPACA1 and GAPDH primer sets generated single, well-defined melt peaks, confirming amplification specificity. No-template controls showed no amplification, and intra-sample technical replicates exhibited low cycle threshold dispersion, supporting data reliability.

Relative SPACA1 expression.

Quantitative analysis using the 2^−ΔΔCt method revealed numerically higher SPACA1 expression in X-sperm (1.023) than Y-sperm (0.506). However, statistical comparison using the Mann-Whitney U test indicated that this difference was not statistically significant (p > 0.05; Fig. 4). Thus, SPACA1 transcript abundance did not differ meaningfully between the two sexed sperm populations under the present experimental conditions.

Fig. 4.

Relative expression of sperm acrosome-associated 1 (SPACA1) in X- and Y-bearing spermatozoa of Bali bulls. Relative mRNA expression was quantified by RT-qPCR and normalized to GAPDH using the 2^−ΔΔCt method. Data are presented as mean ± standard deviation (n = 3 biological replicates). Although SPACA1 expression was numerically higher in X-bearing spermatozoa (1.023) compared to the Y-bearing spermatozoa (0.506), the difference was not statistically significant (Mann-Whitney U test; p > 0.05).

Sensitivity analysis.

Visual inspection of raw cycle threshold values across samples confirmed the absence of outlier amplification profiles. Replicates were consistent, and normalization to GAPDH yielded reproducible patterns. These findings support the biological plausibility of observed expression trends and rule out technical artifacts as confounders.

Discussion

This study investigated whether the SPACA1 gene exhibits differential mRNA expression between X- and Y-bearing spermatozoa in Bali bulls. Despite high post-thaw semen quality and intact RNA profiles, no statistically significant difference was observed in SPACA1 transcript abundance between the two sperm populations. This finding suggests that SPACA1 mRNA expression may not constitute a reliable biomarker for sperm sexing in this breed.

The observed trend, in which X-bearing spermatozoa showed numerically higher SPACA1 levels than Y-bearing counterparts, did not reach statistical significance, being consistent with the known biology of spermatozoa. After meiosis, transcription is largely silenced due to the chromatin compaction and protamine replacement, and the RNA present in ejaculated sperm reflects remnants from earlier spermatogenic stages rather than active transcription.²²

Although several studies have reported the presence of sex-biased RNAs in bovine spermatozoa, the consistency and reproducibility of these findings remain limited due to the methodological variability.²³ However, proteomic approaches offer a more robust strategy. Sex-specific differences in protein abundance related to the motility, metabolism, and membrane organization have been repeatedly identified in sexed semen samples.^10,11 Our result aligns with these observations and underscores that transcript-level evaluation, particularly of SPACA1, may not be sufficiently discriminatory for sexing purposes.

Functionally, SPACA1 encodes a membrane protein essential for acrosome biogenesis and sperm-egg interaction.^9,10 In Bali bulls, deficiency in SPACA1 mRNA and protein has been correlated with poor semen quality and subfertility.¹³ However, our results suggest that while SPACA1 is a potential marker for sperm function, it may not be sex-specific. If sexual dimorphism in SPACA1 expression exists, it is more likely to be observed at the protein level (e.g., sub-cellular localization or surface epitope accessibility) than at the mRNA level.

Technically, this study validated the integrity of extracted RNA, confirmed the specificity of RT-qPCR through melt-curve analysis, and applied standard normalization using GAPDH.^24,25 Although using multiple reference genes could enhance precision, our analyses showed stable cycle threshold values with low variability. Moreover, both X- and Y-bearing sperm fractions yielded intact RNA with clear 28S and 18S bands, minimizing the likelihood of degradation-induced bias.

Limitations of the study include the use of semen from a single bull for each sexed fraction, restricting generalizability and statistical power. Furthermore, the analysis focused only on mRNA without evaluating SPACA1 protein levels, localization, or function. The absence of significant differences does not exclude the possibility of post-transcriptional or translational regulation of SPACA1 between X- and Y-sperms, which may be more effectively captured via proteomic or immunological methods. Finally, our findings are consistent with other investigations suggesting that RNA-based sexing strategies in bulls are less promising than protein-based approaches.²⁶ The potential of SPACA1 lies in its role as a fertility or quality biomarker rather than as a determinant of sperm sex. Recent studies have identified sex-biased sperm surface proteins in cattle, supporting the potential for protein-level discrimination.²³

This study demonstrated that SPACA1 mRNA is detectable in both X- and Y-bearing spermatozoa of Bali bulls. Although a numerical difference in transcript abundance was observed, the variation was not statistically significant. These findings suggest that SPACA1 gene expression at the mRNA level does not serve as a reliable stand-alone biomarker for sperm sexing under current conditions. The data align with the understanding of transcriptional quiescence in mature sperm cells and support the notion that functional or sex-specific differences may be better investigated at the protein level. Further studies are necessary to evaluate SPACA1 protein expression, localization, and immunological accessibility to assess its utility for non-cytometric sexing technologies.

The inability of SPACA1 mRNA to discriminate between X- and Y-bearing spermatozoa in Bali bulls highlights the limitations of transcript-based sexing methods in bovine species. Protein-based strategies, including immunological selection targeting acrosomal or membrane-bound sex-specific markers, represent a more promising avenue. Future research should prioritize high-throughput proteomic profiling, development of monoclonal antibodies against sex-biased surface proteins, and validation in multiple bulls and production lots to enable effective, low-impact sperm sexing suitable for national breeding programs.

Conflict of interest

The authors declare that there is no conflict of interest regarding the publication of this paper.