Sex control of calves is an important strategy in dairy cattle production systems. In Holstein-Friesian (HF) cattle, increasing the proportion of female offspring has direct implications for milk production efficiency and the sustainability of the breeding population (Morek‐kopeć et al., 2021) Reproductive technologies such as artificial insemination (AI) have significantly enhanced animal production efficiency (Ayantoye et al., 2025). The use of sexed semen via AI programs is becoming an increasingly relevant approach to meet the needs of the dairy cattle industry (Quelhas et al., 2023).

However, the use of sexed semen in the field still faces a major challenge: a generally lower fertility rate compared to conventional semen (Pozdyshev et al., 2023); (Sharma et al., 2024). The sexing process can cause mechanical and physical stress that affects sperm integrity and function, reducing viability by approximately 20% (Walsh et al., 2021); (Rasad et al., 2020). These effects are often observed in changes in motility characteristics, especially kinematic parameters.

These parameters perform a key role in sperm transport, interaction with the oocyte, and the success of fertilization. Previous studies have reported that kinematic parameters such as curvilinear velocity (VCL), straight-line velocity (VSL), and beat cross frequency (BCF) are strongly associated with the probability of pregnancy in dairy cattle (Araya-Zúñiga et al., 2024). Large-scale studies show that the pregnancy rate with sexed semen still fluctuates, ranging from 69%–87% in heifers and 72%–85% in lactating cows, compared to conception rate (CR) achieved through conventional semen (Butler et al., 2014); (Maicas et al., 2019).

Percoll density gradient centrifugation (PDGC) offers a simple and economical alternative to flow cytometry for sperm sex determination (Safa et al., 2025). This method separates sperm carrying the X or Y chromosome based on density (Yekti et al., 2023). Evaluating the kinematic characteristics after Percoll separation is important to assessing semen suitability for AI (Tanga et al., 2021). Changes in sperm quality observed in vitro may not predict in vivo fertilization success (Santolaria et al., 2023). One approach to increase pregnancy chances, especially when sperm quality decreases due to separation processes, is the double-dose insemination technique, involving two inseminations during one estrus (Utami et al., 2022). While promising, the effectiveness of this method, particularly with PDGC-sexed semen, remains uncertain in field conditions.

Although sperm sexing techniques, particularly those using the PDGC method, have been extensively developed, information regarding the effects of combining this method with double-dose AI on sperm kinematic quality and reproductive performance remains limited. Previous studies have generally focused only on evaluating semen quality in vitro or in vivo fertility outcomes separately, and the integration of these two approaches has not been widely reported. Furthermore, studies on the effectiveness of PDGC methods in maintaining sperm function while improving reproductive success under field conditions remain incomplete. Therefore, research is needed to comprehensively examine the relationship between the quality of sexed sperm and the resulting reproductive performance. This study examines the kinematic characteristics of sexed semen produced by PDGC and evaluates its fertility performance following double-dose AI during the same estrus cycle in HF cows, with the aim of optimizing the use of sexed semen in dairy farming systems.

The quality of fresh semen was good, with motility exceeding 70%. This reflects good reproductive function in bulls as well as a plasma composition that supports sperm viability (Syarif et al., 2026). A slightly acidic pH contributes to membrane stability and metabolic balanced movement. Balanced ALH values indicate effective propulsion without excessive hyperactivation (Yusuf et al., 2026). These characteristics confirm that the semen is suitable for further processing.

The PDGC method for sexing sperm relies on the principle of separating cells based on differences in density and molecular mass (Rahmawati et al., 2025). Sperm carrying the X chromosome have approximately 3%-4% higher DNA content than sperm carrying the Y chromosome (Pozdyshev et al., 2023). They have a density difference that allows separation through concentration gradients. However, this density difference is relatively small, so the centrifugation process not only selects based on chromosomes but also has the potential to affect the physiological quality of sperm due to mechanical stress and changes in the microenvironment during the separation process.

The results showed that PDGC significantly reduced motility compared to conventional semen. This reduction was likely caused by centrifugal force, which increased mechanical stress on the plasma membrane and flagellum structure. This stress could trigger an increase in the production of reactive oxygen species (ROS) (Aitken, 2017), which is known to cause membrane lipid peroxidation, membrane fluidity disorders, and mitochondrial dysfunction (Abdelnour et al., 2022). Because sperm movement is highly dependent on ATP production through oxidative phosphorylation in the midpiece, mitochondrial dysfunction will directly affect the proportion of cells capable of active movement (Amaral, 2022)). However, motility in the PDGC group remains within a range suitable for fertilization.

The results of this study align with (Safa et al., 2025), showing that density gradient based sexing can preserve sperm motility. However, there are differences compared to (Syarif et al., 2026), who reported higher motility, likely due to variations in the sexing protocol, specifically the use of the albumin sedimentation method, the type of diluent, and the freezing process.

These differences underscore that the method’s effectiveness depends on the technical conditions used.

DAP represents the average length of the path, DSL describes the straight-line distance traveled, and DCL indicates the total curved path (O’Meara et al., 2022); (Horst, 2020). The increase in these three parameters in G1 indicates longer, more active trajectories, reflecting more optimal flagellar propulsion. This showed that a 20%-60% gradient is more effective at maintaining sperm with good translational motility. Conversely, a decrease in G2 values (20%-65%) indicates that a higher density gradient may disrupt cellular structural integrity or energy function, leading to shorter, less dynamic trajectories (Figure 2). The PCA results indicate that treatments that enhance speed parameters tend to produce sperm with more dynamic movement, whereas treatments related to linearity parameters promote more directed movement. The balance between these two aspects is a key factor in determining sperm fertility potential (Syarif et al., 2026).

Significant increases in VCL and ALH, especially in the PDGC 20%-60% group, indicate a change in sperm motility, making it more dynamic and energetic. High VCL reflects high actual trajectory speed, while ALH describes increased amplitude of lateral head movement. The combination of these two parameters is physiologically associated with a pattern of hyperactivation, a change in motility that occurs during the capacitation process in the female reproductive tract. Molecularly, hyperactivation is associated with increased intracellular Ca²⁺ flux and activation of cAMP–PKA signaling pathways during capacitation(Cordero-Martínez et al., 2022). This movement pattern plays an important role in penetrating the zona pellucida and achieving successful fertilization. Therefore, the increase in these parameters may reflect the selection of a subpopulation of sperm with greater metabolic and energy capacity during the PDGC process. However, hyperactivation that occurs too early can also reduce sperm viability before they reach the fertilization site (Zafar et al., 2021). The absence of a decrease in reproductive performance indicates that these changes are still within adaptive physiological limits.

Changes in LIN and WOB, which decreased in the PDGC 20%-60% group, indicate a change in movement pattern towards hyperactivation. A low LIN indicates a less straight trajectory, while a decreased WOB value reflects an imbalance between average velocity and curvilinear velocity (Rotar et al., 2026); (Wahyudi et al., 2025). This pattern aligns with the movement pattern required when approaching the oocyte. However, if hyperactivation occurs too early due to the pressure from the sex determination process, sperm may lose energy before reaching the fertilization site. The parameters of progressive motility, VAP, STR, and BCF did not show significant differences. This indicates that although changes in certain movement dynamics, the ability of sperm to maintain progressive movement direction and flagellar beat frequency remained stable. The PDGC sex determination method does not disrupt the overall control of flagellar movement coordination. This stability is important because fertilization requires a balance between speed, direction of movement, and metabolic endurance (Zhang & Xu, 2025).

Although several motion parameters showed significant changes, analysis of NRR1, NRR2, CR, and CvR showed no significant differences between treatments. This indicates that the changes that occurred at the kinematic level were not large enough to interfere with the in vivo fertilization process. Successful fertilization requires sperm with intact DNA, acrosome stability, and the ability to activate oocytes by releasing factors such as phospholipase C zeta (PLCζ), which triggers cytoplasmic Ca²⁺ oscillations (Gonzalez-Castro & Carnevale, 2023). If PDGC causes severe chromatin damage or acrosomal disruption, a decrease in pregnancy rates or an increase in early embryo loss should be observed. Since this did not occur, it can be concluded that the structural and molecular integrity of sperm is maintained.

One factor that contributes to maintaining reproductive performance in sexed semen groups is the use of the double-dose method during AI. Sexed semen typically has a lower concentration of viable sperm due to the selection process and potential cell loss during centrifugation (Barros Mothé et al., 2018); (Priyanto et al., 2023). The use of double-dose AI aims to compensate for the potential reduction in the number of viable sperm reaching the fertilization site (the ampulla of the oviduct). By increasing the number of cells inseminated, the probability of interaction between viable sperm and the oocytes is maintained. This strategy aligns with the principle that fertilization success is determined not only by the quality of individual sperm but also by the number of cells that can survive, migrate, and achieve capacitation on time within the reproductive tract (Hoek et al., 2022).

Physiologically, only a small fraction of sperm successfully reach the site of fertilization due to natural selection in the cervix, uterus, and fallopian tubes. Therefore, the double-dose approach is rational to minimize the risk of fertilization failure by reducing the initial sperm population following the sexing process. The fact that reproductive performance does not differ significantly indicates that the combination of the PDGC method and the double-dose strategy maintains a balance between the quality and quantity of sperm required to achieve optimal reproductive performance.

The discussion of sex ratios yields additional strategic insights from a production management perspective. Figure 3 showed that sexed semen groups produced a higher proportion of female calves than conventional semen. While male calves still dominate in the control group, the sexing group showed a significant shift towards female calf dominance (around 67%-70%). The results of this study show a higher proportion of female calves born compared to that reported by Mendes et al. (2022) who reported a percentage of 47.1% female and 52.9% male calves in insemination using sexed semen, and 51.3% female and 48.7% male calves in the use of conventional semen. In addition, (Shabirah et al., 2025) reported a 85.2% proportion of female calves. This confirms that the density-based PDGC method can increase the chances of obtaining offspring of the desired sex, even though the success rate does not reach 100%.

The increase in the proportion of female calves reflects relative success in enriching X chromosome-carrying sperm in certain gradient fractions. Although the difference in DNA content between the X and Y chromosomes is relatively small, the multi-stage centrifugation process allows for the distribution of fractions with dominance of one population. Thus, although this method is not as precise as flow cytometry-based technology, PDGC still demonstrates practical effectiveness in significantly increasing the chances of female births compared to conventional semen, which naturally produces a ratio close to 50:50. However, this study has several limitations, particularly regarding the relatively small sample size and field conditions that could potentially influence fertility outcomes. Therefore, future studies are recommended to use a larger sample size and more controlled conditions to enhance the validity of the results.

Sexing sperm using the PDGC method significantly alters several kinematic variables, including motility, DAP, DCL, DSL, VSL, VCL, LIN, ALH, and WOB, suggesting changes in movement dynamics driven by the density-based separation process. However, these changes do not reduce in vivo reproductive performance, as NRR, CR, and CvR values remain comparable to those of conventional semen, especially when a double-dose insemination strategy is used to compensate for a potential reduction in viable sperm. The use of sexed semen increased the proportion of female offspring compared to conventional semen, indicating the effectiveness of this method in enriching X chromosome-carrying sperm. Overall, PDGC-based sexing represents a practical and relatively safe approach to increase the chances of female offspring.

The authors declare that there are no financial or commercial relationships that could be construed as a potential conflict of interest in relation to this research.

The authors gratefully acknowledge the financial support provided by the Indonesia Endowment Fund for Education (Lembaga Pengelola Dana Pendidikan/LPDP), Grant/Award Number: LOG-10860/LPDP.3/2024. The authors would also like to express their sincere gratitude to the Singosari National Artificial Insemination Center for granting access to laboratory facilities and for the technical assistance that significantly contributed to the completion of this research.

During the writing process, the authors used Grammarly software to assist with grammar and sentence structure editing. All edits have been reviewed by the author, who is responsible for the final content of the published manuscript.