Indonesia still relies on imports for 40% of its national beef demand(Secretariat, 2023). This dependence on imports increases the vulnerability of national food security(Kusumaningrum et al., 2024). Therefore, strategic solutions are required, including efforts to enhance reproductive efficiency through the application of reproductive technologies, such as spermatozoa sexing. Spermatozoa sexing enables the selection of the desired sex of offspring, thereby optimising livestock productivity. The process of spermatozoa sexing relies on distinguishing spermatozoa that contain different sex chromosomes(Saputro et al., 2022). There are variations in size, shape, weight, density, motility, charge, and surface biochemical composition between X and Y-chromosome-bearing spermatozoa(Garner & Hafez, 2000).

Limousin cattle are highly desired by farmers in Indonesia. This breed, which originates from France, has been selectively developed to enhance superior traits(Mariadassou et al., 2020). They have several advantages, including adaptability to humid environments in tropical areas such as Indonesia(Nurhidayat et al., 2024), as well as high feed efficiency and carcass yield(Kayar & İnal, 2022)

One of the spermatozoa sexing techniques used in Indonesia is the albumin sedimentation method, which utilises bovine serum albumin (BSA). The effectiveness of this method can vary depending on specific conditions and protocols, but it is relatively straightforward(Pinto-Pinho et al., 2023). In beef cattle, especially Limousin cattle, the quality of semen produced using this method has not been evaluated at the molecular level. Semen quality encompasses not only spermatozoa volume, concentration, and motility but also molecular characteristics, including DNA integrity and spermatozoa kinematics. Research on the impact of spermatozoa sexing on DNA fragmentation and spermatozoa kinematics in Indonesia is still limited. Currently, no research has specifically evaluated this technique in Limousin cattle in Indonesia, making this the first study of its kind. Therefore, this study aimed to assess the effects of spermatozoa sexing using the albumin sedimentation method on semen quality, DNA fragmentation, and spermatozoa kinematics in Limousin cattle.

The progressive and total motility of fresh semen averaged more than 80%, and the viability values for all bulls also exceeded the standard threshold of 80%, indicating that semen was suitable for further processing(Safa et al., 2025). The plasma membrane integrity exhibited greater values than motility, as reported by Baharun et al. (2023)(Baharun et al., 2023), suggesting that the spermatozoa are capable of optimal functionality. Macroscopic evaluation showed that the average semen volume, colour, and pH values were within the normal range, as reported by Royan et al. (2021)(Royan et al., 2021).

The quality of frozen sexed and non-sexed semen, such as total motility, viability (Figure 1A), and plasma membrane integrity (Figure 1B), decreased after freezing due to physical damage, cold shock, mitochondrial dysfunction, and oxidative stress(Hai et al., 2024). Spermatozoa undergo dehydration during freezing, which may reduce the intracellular ice formation(Hine et al., 2019). However, dehydration can alter the structure and function of the plasma membrane, leading to the formation of micropores and a loss of semipermeability(Sun et al., 2020). The quality of frozen sexed and non-sexed semen did not show significant differences, in contrast to the findings of Nurgina et al. (2024)(Nurgina et al., 2024), who reported significant differences in semen quality between sexed and non-sexed semen. These differences may be due to the conventional albumin sedimentation method(Yata, 2022), indicating that it remains highly dependent on the protocol and procedures.

Sexed and non-sexed frozen showed no significant differences in DNA fragmentation (Figure 2A) and acrosome integrity (Figure 2B, Table 3), because the BSA sedimentation method allows high-quality spermatozoa to pass through the gradient, while low-quality spermatozoa remain in the upper layer(Mundana et al., 2023). Al-Kass et al. (2025)(Al-Kass et al., 2025) reported that centrifugation at 1800 rpm for 10 minutes did not reduce spermatozoa quality. This finding is consistent with that of Masrizal et al. (2024)(Masrizal et al., 2024), who reported that spermatozoa sexing using the BSA method did not lead to increased DNA fragmentation. Meanwhile, the flow cytometry method reported by Silva et al. (2016)(Silva et al., 2016) revealed genetic material damage resulting from DNA staining and laser exposure. However, the centrifugation process can increase the production of reactive oxygen species (ROS), which can trigger DNA fragmentation due to damage to the plasma membrane(Dasrul & Harris, 2012). Spermatozoa DNA damage can interfere with pronuclear fusion during fertilisation, causing embryo death(Priyanto et al., 2015).

Acrosome integrity demonstrated stability between sexed and non-sexed semen, due to its higher lipid and protein composition compared to the plasma membrane. Lipids in the acrosome can increase its resistance to lipid peroxidation, which often damages the plasma membrane of spermatozoa(Nofa et al., 2017). Additionally, the structural integrity of the acrosome is maintained during cryopreservation by integral proteins, including protease enzymes(Ferrer et al., 2012). Furthermore, acrosome resistance is significant to ensure that the acrosome reaction occurs in a timely and effective manner during fertilisation(Bernecic et al., 2021).

Progressive motility and spermatozoa kinematics showed significant differences between fresh semen and frozen semen, likely due to mitochondrial damage incurred during the freezing process(Pezo et al., 2021). Mitochondria in spermatozoa produce adenosine triphosphate (ATP), which is the primary source of energy for spermatozoa(Pezo et al., 2021). Mitochondria are responsible for the generation of adenosine triphosphate (ATP), which serves as the primary energy source for spermatozoa(Nofa et al., 2017). Damage to spermatozoa mitochondria can reduce motility, so that good spermatozoa can adapt to the remaining extreme conditions. Progressive motility did not show significant differences between sexed and non-sexed frozen semen. Safa et al. (2025)(Safa et al., 2025) also reported no significant difference in progressive motility between sexed and non-sexed frozen semen.

The differences in distance parameters (DCL, DAP, and DSL) between fresh and frozen semen may be due to changes in spermatozoa membrane fluidity and cytoplasm consistency during freezing(Grötter et al., 2019), which can affect spermatozoa flagella motility. On the other hand, the effect of spermatozoa sexing did not show significant differences in distance parameters. Fertilisation success indicators can be determined by DCL, DAP, and DSL values(Utami et al., 2025). The overall spermatozoa trajectory is indicated by the DCL value(Víquez et al., 2020). The average distance travelled by spermatozoa along the trajectory is indicated by the DAP value(Sinha et al., 2021). The DSL value indicates the minimum distance between the starting and ending points of movement(Foutouhi et al., 2023). The ability of spermatozoa to penetrate the oocyte layer with sufficient speed and movement is crucial for successful penetration(Tumova et al., 2021).

The velocity parameters (VSL, VCL, and VAP) differ significantly between fresh and frozen semen, which can be attributed to mitochondrial damage during the freezing process, affecting flagellar activity and overall spermatozoa motility(Utami et al., 2025). In contrast, no significant differences were observed among non-sexed, sexed X, and sexed-Y frozen semen. These results are in line with the study by Safa et al. (2025)(Safa et al., 2025), which reported no significant differences in frozen semen spermatozoa velocity parameters after the sexing process. Spermatozoa 275 of fresh and frozen semen showed values above the minimum VSL threshold of 25 µm/s(Maulana et al., 2021). The VCL values obtained in this study were greater than 90 µm/s, which is categorised as fast(Křížková et al., 2017). The parameters VCL, VSL, and VAP have been proven to be effective predictors of fertility(Maulana & Said, 2019). Specifically, VCL and VSL are directly related to fertilisation capacity, while VAP has been shown to have a strong correlation with pregnancy success(Nagy et al., 2015).

Significant differences in STR WOB values between fresh and frozen semen may be due to cold stress. Cold stress on spermatozoa is known to affect the plasma membrane, increasing ion permeability, which primarily causes the influx of calcium ions (Ca²⁺) into the spermatozoa cytosol(Treulen et al., 2018). which in turn affects spermatozoa motility patterns. In this study, semen sexed-Y had higher LIN values compared to semen non-sexed, indicating that semen containing a higher proportion of Y-chromosome-bearing spermatozoa can move more linearly. Daloglu et al. (2018)(Daloglu et al., 2018) reported that the linearity (LIN) of Y-chromosome-bearing spermatozoa consistently showed higher values in trajectory. The identification of hyperactive spermatozoa that move quickly and strongly, but not progressively and linearly, can be explained by STR and LIN values(Hansen et al., 2006). Spermatozoa with STR values greater than 50% and LIN higher than 35% are categorised as spermatozoa that move linearly(O’Meara et al., 2022). Sexed and non-sexed semen in this study had STR values above 50% and LIN exceeding 35%, indicating that the sexing using the albumin sedimentation method can maintain linear movement. The WOB values in this study were higher than those reported by Utami et al. (2025)(Utami et al., 2025). The actual oscillatory movement along the trajectory can be demonstrated by the WOB value(Valverde et al., 2019). The balance between stability and flexibility of spermatozoa movement, as indicated by a high WOB value, is the key to successful fertilisation(Utami et al., 2025).

Fresh and frozen semen exhibited different ALH and BCF values, indicating alterations in spermatozoa head motility patterns resulting from the freezing and thawing process. Factors such as increased intracellular calcium ions, changes in membrane flexibility, and cold stress during freezing can affect these motility patterns(Afriani et al., 2024). In addition, sexed-Y frozen semen showed different ALH and BCF values compared to non-sexed and sexed-X semen, which may be related to the spermatozoa sexing procedure. During this process, spermatozoa undergo physical and chemical manipulation that affects spermatozoa head movement patterns(Sringarm et al., 2022). The results of this study indicate that sexed-Y semen exhibits higher BCF and lower ALH values than the other groups, consistent with the findings of Afriani et al. (2024)(Afriani et al., 2024). This variation is likely associated with the genetic content of the spermatozoa, as X-chromosome-bearing spermatozoa contain more DNA. In contrast, Y-chromosome-bearing spermatozoa have higher levels of miRNA and tsRNA(Zhou et al., 2020). These differences may explain the relatively higher motility efficiency observed in Y-chromosome-bearing spermatozoa.

The ALH values of frozen sexed and nonsexed semen were in the range of 4–6 µm. The ideal ALH values for increasing fertility potential range from 2.5 to 6.5 µm(Belala et al., 2019). indicating that the spermatozoa in this study can support successful fertilisation. The observed BCF values were higher than those reported by Prastiya et al. (2023)(Prastiya et al., 2023). Higher BCF reflects more stable and regular motility patterns(Ratnawati et al., 2020) as well as greater energy expenditure by spermatozoa(Vigolo et al., 2022). Moreover, Y-chromosome-bearing spermatozoa exhibited higher BCF values compared to X-chromosome-bearing spermatozoa and non-sexed semen, indicating a more stable and regular motility pattern. It should be noted, however, that this study has certain limitations, as only frozen sexed and non-sexed semen were evaluated, without including semen before freezing. Nevertheless, these findings provide valuable insight into the effects of spermatozoa sexing on semen quality and spermatozoa kinematics in Limousin cattle for artificial insemination.

Sexing spermatozoa using the BSA method does not affect spermatozoa quality in Limousin bulls. The kinematic profile, characterised by lower ALH and higher BCF values in sexed-Y, indicates that Y-chromosome-bearing spermatozoa have a more stable and regular movement pattern, which may provide better fertilisation capacity compared to X-chromosome-bearing spermatozoa and non-sexed spermatozoa. These findings imply that spermatozoa sexing using the BSA method can be used in artificial insemination without compromising spermatozoa quality and may support improved fertilisation efficiency and sex control in cattle breeding.