Meiosis is a specialized reductional process of genome segregation essential for sexual reproduction, producing haploid gametes from diploid germ cells. It comprises a series of tightly coordinated events, including homologous chromosome pairing, synapsis, recombination and segregation, which together ensure genetic diversity and maintain chromosome number across generations (Yang et al., 2026). In mammals, meiotic regulation is highly dynamic and differs markedly between males and females (Hu et al., 2026). In females, germ cells begin meiosis during foetal development and remain arrested at the diplotene stage of prophase-I until ovulation. Whereas, in male germ cells, meiosis is initiated after birth as part of the continuous process of spermatogenesis. These differences highlight distinct regulatory mechanisms governing germ cell development in the two sexes (Saitou et al., 2026). Although much of the current understanding of meiotic regulation derives from model organisms such as mice and humans, similar fundamental mechanisms are conserved in livestock species, including buffalo.
       
The initiation of meiosis is regulated by a coordinated network of signalling pathways that drive the transition of germ cells from mitosis to a meiotic program. One of the key regulators of this process is STRA8 (Stimulated by Retinoic Acid 8), whose expression is triggered by retinoic acid (RA), a critical signal for the onset of meiosis (Zhao et al., 2024). Along with RA, additional pathways such as BMP signalling also contribute to the regulation of meiotic entry and progression (Zhang et al., 2022). The expression of meiotic genes also shows clear differences between the testis and the ovary. In the foetal ovary, STRA8 expression occurs in a relatively synchronised manner, leading to a coordinated process of meiotic entry. In contrast, in the testis, STRA8 expression is restricted to specific subsets of spermatogonia and exhibits a cyclical, asynchronous pattern (Cheung et al., 2025). Although these mechanisms are well studied in model organisms, they are thought to be largely conserved in buffalo, though their precise regulation remains unclear. Once meiosis begins, early prophase-I involves proteins that organize chromosomes and initiate recombination. HORMAD1, the first responders, bind to unsynapsed chromosome regions to promote DNA double-strand breaks and facilitate correct homolog pairing through checkpoint regulation (Raveendran et al., 2026). Simultaneously, recombination proteins such as DMC facilitate strand exchange and DNA repair during these initial stages (Cheng et al., 2026).
       
As meiosis advances, structural proteins become crucial for proper chromosome alignment and organization. SYCP1, SYCP2 and SYCP3 are important proteins that help chromosomes pair correctly during meiosis. SYCP2 and SYCP3 act early in the process, particularly during the leptotene stage, when they attach to the chromosomes and form a basic structural framework called the axial elements (Adams and Davies, 2023). This framework helps organise DNA and keep chromosomes stable and properly aligned. As meiosis progresses to the zygotene stage, SYCP1 comes into play, acting as a connector that forms transverse filaments between paired chromosomes (Guo et al., 2025). By the pachytene stage, this connection becomes complete, allowing the chromosomes to remain closely paired, which is necessary for proper recombination and exchange of genetic material. SYCP3 is vital for forming the synaptonemal complex, enabling homologous chromosomes to align and synapse properly (Cesar and Kim, 2026), while REC8, a meiosis-specific protein, maintains sister chromatid cohesion, which is essential for accurate segregation (Liu et al., 2025). In later stages, especially during pachytene, crossover events are completed with the assistance of MLH1 and MLH3, which stabilise recombination sites and promote correct chromosome segregation (Payero and Alani, 2025). Any disruption in the coordinated expression or function of these genes can affect normal meiotic progression and may lead to infertility, which is particularly important in livestock species such as buffalo, where reproductive efficiency directly impacts productivity (Singh et al., 2023).
       
Understanding these sex-specific differences is particularly important in buffalo, where both male fertility (semen quality) and female reproductive performance (oocyte quality) are critical to breeding success (Morgan et al., 2020). Recent advances in molecular techniques, particularly quantitative gene expression and single-cell RNA sequencing, have enabled high-precision study of gene expression patterns (Wang et al., 2023). In buffalo and closely related bovine species, transcriptomic studies are beginning to elucidate the molecular landscape of germ cell development and meiotic progression, offering valuable insights into folliculogenesis, spermatogenesis and oocyte competence (Xu et al., 2024). Considering the economic and biological importance of buffalo reproduction, a deeper understanding of meiosis-specific gene expression in buffalo gonads is essential. However, compared with well-studied model organisms, information on comparative expression patterns of key meiotic genes between buffalo testis and ovary remains limited. As Meiosis is a fundamental process in both male and female germ cells, although its progression differs between the testis and the ovary. Comparing the relative expression of selected meiotic genes, including HORMAD1, STRA8, SYCP3, REC8, MLH1, DMC and SYCP, in buffalo testis and ovary using quantitative PCR, provides information on their tissue-specific expression patterns. Therefore, the present study aims to analyze the expression profiles of selected meiosis-specific genes. This will help to elucidate sex-specific differences in meiotic regulation and contribute to a better understanding of germ cell development in buffalo.

Collection and preparation of the ovary and testis samples
 
Buffalo ovarian and testicular tissues (n=3) were collected from slaughterhouse-based animals of approximately 3 years of age. Cyclic ovaries were selected based on the presence of follicles and corpus luteum. Testes were collected from sexually mature animals with normal morphology and active spermatogenesis and were transported on ice packs. The tissue surface was sterilized with 70% ethanol and washed 4-6 times with DPBS containing 50 μg/mL gentamicin sulphate to remove contaminants. The ovarian cortex was carefully dissected and chopped into small pieces; this region contains follicles at various developmental stages and stromal cells. In the testis, the tunica albuginea was removed and the tissue was further dissected to isolate the seminiferous tubules, which are the primary sites of meiosis. The processed tissue samples were immediately homogenized in liquid nitrogen and stored in TRIzol until further molecular analysis. The work was conducted at the ICAR-National Dairy Research Institute, Karnal.
 
RNA isolation and cDNA preparation
 
Total RNA was isolated from ovarian and testicular samples (n=3) using TRIzol. Chloroform and isopropanol were then added to the tube to precipitate nucleic acids and proteins (Shavi et al., 2025). The final wash was performed with 70% ethanol, followed by centrifugation to remove residual ethanol. The pellet was dried for 15-20 minutes and eluted in 20 μL of nuclease-free water. For cDNA synthesis, RNA concentration was measured and adjusted to 100 ng/μL for the ovary and testis samples. cDNA was synthesized using the RevertAidTM First Strand cDNA synthesis kit (Fermentas, Life Sciences, USA) according to the manufacturer’s instructions. For cDNA synthesis, 1 μL of total RNA (100 ng), 2 μL dNTP mix (10 mM), 1 μL oligo(dT) (10 μM), 4 μL reaction buffer, 1 μL RibolockTM RNase inhibitor (20 u/μL), 1 μL RevertAidTM M-MuLV Reverse Transcriptase (200 u/μL) and nuclease-free water were added to make a volume of 20 μL. First, the reaction mixture containing RNA, primer and nuclease-free water was incubated at 65°C for 5 min in a thermal cycler. After that, the reaction mixture was chilled on ice for 5 min and the remaining components were added and the mixture was incubated at 42°C for 60 min. The synthesized cDNA was stored at -20°C until use for qPCR. The quality of the synthesized cDNA was evaluated by amplification of the housekeeping gene GAPDH using conventional PCR. Amplification of the meiosis-specific genes (Table 1) was also performed using conventional PCR. The PCR reaction was carried out in a total volume of 10 µL containing cDNA template (1 µL), gene-specific primer (10 mM), dNTPs, Taq DNA polymerase, reaction buffer and nuclease-free water. The amplification conditions included an initial denaturation at 95°C, followed by 40 cycles of denaturation at 95°C, annealing at 60°C and extension at 72°C, with a final extension at 72°C. The PCR products were analyzed by agarose gel electrophoresis.


 
Quantitative gene expression
 
Relative quantification of mRNA for multiple genes was performed using the CFX96 real-time system (Bio-Rad, Hercules, USA). GAPDH was used as a reference gene for all experiments. The qPCR reactions were carried out using Maxima SYBR Green qPCR Master Mix (2X) with a separate ROX (Thermo Fisher Scientific Inc., USA). Each run was performed in duplicate in a 10 μL reaction volume containing 5 μL of fluorescence dye, 1 μL of gene-specific primers (forward and reverse, 10 μM) and 1 μL of template cDNA. The final volume was adjusted with nuclease-free water. The PCR conditions were as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s (Tripathi et al., 2024; 2025). The melting cycle ranged from 65°C to 95°C with a transition rate of 0.5°C/s. qPCR specificity was confirmed by analyzing the melting curves generated using CFX Manager Software. Relative gene expression was calculated using the 2^ΔΔCt method (Livak and Schmittgen, 2001). Gene-specific primers were designed to amplify fragments of approximately 150-200 bp (Table 1), preferably towards the 3′ end of the cDNA. Primer design was performed using conserved regions of bovine or buffalo sequences with Primer3 software (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3-www.cgi). The MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines were followed throughout the study to ensure the accuracy, reliability and reproducibility of the qPCR data.
 
Statistical analysis
 
Statistical analysis was performed using GraphPad Prism 7 software. Gene expression analysis was performed using the comparative Ct (2^ΔΔCt) method after normalization with the housekeeping gene. Relative fold-change values were calculated with respect to the control group. Data are presented as mean ± SEM from independent biological replicates. Statistical significance among groups was analyzed by Student’s t-test and one-way ANOVA. Differences were considered significant at P<0.05.

RNA isolation from the ovary and testes tissue
 
The total RNA was successfully isolated from all samples and the quality and quantity were assessed by spectrophotometric analysis, which showed A260/A280 ratios of 1.94±0.02 and 2.08±0.03 in the ovary and testis samples, respectively. The total RNA concentrations in the ovary and testis samples were 1856 ng/µL and 2567 ng/µL, respectively. The A260/A230 ratios were 2.0 and 2.2, confirming negligible contamination by phenol, salts, or other organic compounds. These values indicate that the isolated RNA was sufficiently pure for downstream molecular applications.
 
cDNA synthesis and amplification of the meiosis-related genes
 
Complementary DNA (cDNA) was synthesized from high-quality RNA samples and its integrity was verified by amplification of the housekeeping gene GAPDH. Agarose gel electrophoresis of the PCR products revealed a clear and specific band of the expected size (176 bp) (Fig 1), confirming successful cDNA synthesis. Subsequently, the meiosis-specific genes HORMAD1, STRA8, REC8, MLH1, DMC, SYCP1, SYCP2 and SYCP3 were amplified in both testicular (Fig 1) and ovarian (Fig 2) tissues. Agarose gel electrophoresis showed distinct bands corresponding to the expected sizes for all selected genes, confirming their expression in both tissues and validating their suitability for downstream gene expression analysis.




 
Quantitative gene expression of meiosis-related genes in ovary and testis
 
The qPCR revealed that Ct values in ovary samples ranged from 26.21 to 29.44, indicating relatively limited variation among the genes analysed. SYCP3 and REC8 showed the lowest Ct values (26.21), suggesting comparatively higher expression, whereas SYCP1 exhibited the highest Ct value (29.44), indicating lower expression (Table 2). Overall, the variation within the ovaries was about 3 cycles, reflecting minor differences in gene expression levels. By contrast, in testicular samples, Ct values ranged from 14.50 to 19.18, indicating greater variation than in the ovary. The lowest Ct value was observed for HORMAD1 (14.50), suggesting the highest expression, while REC8 (19.18) showed relatively lower expression among the analyzed genes. The variation within the testis was approximately 4-5 cycles, indicating greater differences in gene expression levels. Overall, gene expression variability was more pronounced in the testis than in the ovary (Table 2).


 
Comparative meiosis-related gene expression in ovary and testis
 
The relative mRNA expression of STRA8 was assessed in ovary and testis samples and a significantly higher (P<0.05) expression was observed in the testis compared to the ovarian tissues (123.15±13.19 vs 1.00±0.00 folds, Fig 3), indicating a strong tissue-specific expression pattern, suggesting an important role of STRA8 in the initiation of meiosis, particularly in the testis. The relative mRNA expression of HORMAD1 was significantly higher (P<0.05) in the testis compared to the ovary (751.16±82.15 vs 1.00±0.00 folds, Fig 3). These results indicate a strong tissue-specific expression pattern, suggesting a potential role of HORMAD1 in meiotic processes.


       
Likewise, the relative mRNA expression of DMC was evaluated, showing 78.86±8.86-fold higher (P<0.05) in testicular tissue compared to ovarian tissue (Fig 3). This pronounced difference highlights a tissue-specific expression pattern and underscores the essential role DMC plays in promoting homologous recombination by facilitating strand invasion and repair of DNA double-strand breaks during early meiotic segregation. In comparison to the ovary, the testis exhibited a substantial increase in the relative mRNA expression of REC8 (P<0.05). The expression in testicular tissue was elevated by 8.17±0.60 fold (Fig 3). The synaptonemal complex genes SYCP1, SYCP2 and SYCP3 showed significantly higher (P<0.05) expression levels of 79.02±7.05, 87.48±1.90 and 45.20±4.96, respectively, compared to the ovary (Fig 3). The synaptonemal complex plays a vital role in facilitating homologous recombination and crossover events, thereby ensuring accurate chromosome segregation. Its proper assembly and disassembly are crucial for the normal progression of meiosis and overall fertility.
       
A marked increase in the relative mRNA expression of MLH1 and REC8 was observed, with 97.11±8.42- and 8.17±0.60-fold upregulation (P<0.05) (Fig 3), respectively, in the testis compared to the ovary. This differential expression pattern highlights the tissue-specific nature of MLH1 and REC8, supporting their critical role in forming crossovers between homologous chromosomes and ensuring proper chromosome segregation. These observations indicate relatively higher expression of meiosis-associated genes in the testis than in the ovary.
       
The present study showed that meiosis-specific genes STRA8, HORMAD1, DMC, SYCP1, SYCP2, SYCP3, REC8 and MLH1 had Ct values of 26.21 to 29.44 in ovarian samples, indicating relatively small fold changes and minor differences in gene expression. By contrast, in testicular samples, Ct values for these genes ranged from 14.50 to 19.18, indicating greater variation than in the ovary. Overall, gene expression variability was more pronounced in the testis than in the ovary. When expression of these genes was compared between the two tissue types, it was significantly higher in testicular tissue than in ovarian tissue. This difference can be attributed to the fundamental differences in gametogenesis between males and females. In the testis, spermatogenesis is a continuous process and meiosis occurs actively throughout adult life. In contrast, in the ovary, oocytes enter meiosis during foetal development and remain arrested for long periods, resulting in comparatively lower expression of meiotic genes. These findings are supported by recent studies in bovines. Single-cell transcriptomic analysis of bovine testicular tissue has shown that meiotic genes such as SYCP3 are specifically upregulated during the transition from spermatogonia to spermatocytes, marking the initiation of meiosis and active germ cell differentiation (Jin et al., 2026). This stage-specific expression pattern highlights the dynamic nature of spermatogenesis and explains the elevated gene expression observed in testicular tissue. Similarly, studies in cattle and yak have demonstrated that genes such as DMC1 and SYCP3 are closely associated with normal meiotic progression and reduced expression of these genes leads to meiotic arrest and infertility (Cao et al., 2022).
       
Further evidence indicates that recombination-related genes, including DMC1 and RAD51, are actively expressed during meiotic prophase in bovine germ cells, emphasising their role in homologous recombination and the maintenance of chromosomal integrity during spermatogenesis (Robert et al., 2021). Earlier studies have also reported that key meiotic genes, such as STRA8, SYCP3 and DMC1, are tightly regulated and exhibit stage-specific expression, with higher levels in actively dividing germ cells (Ishiguro et al., 2020). In mammals, proteins involved in recombination and synapsis, including DMC1 and synaptonemal complex components (SYCP1, SYCP3), are essential for proper chromosome pairing and recombination during meiosis (Zhang et al., 2019). In addition, activation of meiosis is regulated by factors such as STRA8, which initiates downstream meiotic gene expression during spermatogenesis (Zhang et al., 2021). Comparative studies across mammals have also shown that, whereas meiotic gene expression in the ovary is temporally restricted, sustained expression in the testis is associated with continuous germ cell renewal (Maroto et al., 2025). Conserved expression of markers such as SYCP3 and DMC1 across species further supports their role in active germ cell differentiation, particularly in the testis (Houmard et al., 2009).
       
The lower expression of these genes in ovarian tissue observed in the present study can be explained by the timing of meiosis in females. In mammals, including bovines, oocyte meiosis begins during foetal life and is arrested at the diplotene stage, resulting in reduced transcriptional activity of meiotic genes in adult ovaries. Recent studies also support this sex-specific pattern, showing that genes involved in recombination and synapsis are expressed during early meiotic stages in both sexes but remain more active only in the testis due to continuous spermatogenesis (Ruan et al., 2026). Studies in mouse models further demonstrate that disruption of meiotic gene regulation leads to defective chromosome pairing and infertility, underscoring its critical role in germ cell development (Feng et al., 2022). The differential expression of the selected meiotic genes observed between buffalo testis and ovary is consistent with previous studies demonstrating sex-specific expression patterns during mammalian gametogenesis. Rockenbach et al., (2023) reported that several meiosis-associated genes, including HORMAD1, SYCP3 and other synaptonemal complex-related genes, exhibit differential expression between male and female gonads, reflecting the distinct molecular regulation of spermatogenesis and oogenesis. Similarly, HORMAD1 has been shown to play an essential role in homologous chromosome synapsis, meiotic recombination and chromosome segregation and its disruption leads to defective meiotic progression and infertility in mammals (Shin et al., 2010). The expression patterns observed in the present study, therefore, support the conserved role of these meiotic genes during germ cell development and provide baseline information on their relative expression in buffalo reproductive tissues. Overall, the results of the present study are consistent with previous findings reported in bovine and other mammalian species.  These differential expression patterns highlight the sex-specific regulation of meiosis and germ cell development in buffalo. The comparative analysis of the relative expression of selected meiotic genes in testis and ovary revealed tissue-specific expression patterns. These differences may reflect the distinct physiological processes of spermatogenesis and oogenesis. Although only a limited number of meiotic genes were examined, the findings provide useful baseline information on their expression during normal gametogenesis in buffalo. They may serve as a reference for future studies investigating reproductive function and fertility.

The study revealed significantly higher expression of meiosis-specific genes in testicular tissue compared to ovarian tissue, reflecting continuous spermatogenesis in males, where meiosis is ongoing, whereas in females, meiosis initiates during foetal development and remains arrested, leading to reduced gene expression in adult ovaries. This upregulation of meiotic and recombination-related genes during gametogenesis confirms these genes as reliable markers of active meiosis.
 
Ethical consideration
 
Since the sample was collected from a slaughterhouse, no ethical approval is needed.

It is declared that there is no conflict of interest related to the authorship, research, or publication of this manuscript.