The industrial production of sexed semen has been a breakthrough in cattle breeding. Sexed semen is fully applied in the development of excellent bulls and the timely replacement or expansion of herds. The production of cattle greatly benefits from the use of sexed semen. It lowers the cost of progeny testing programs and embryo transfer, for example, when producing desired-sex calves for the beef or dairy industries [1]. Additionally, pandemic diseases are the main problems affecting livestock production through many animal deaths, especially endemic diseases (i.e., foot and mouth disease), which restrict international animal trade, and emerging diseases (i.e., lumpy skin disease) that threaten and cause economic losses in the cattle sector [2,3]. Most notably, the aforementioned epidemic has resulted in a diminished reproductive capacity among the remaining female population, impacting the expansion of the overall population. The most well-known invention that enhances the number of female offspring is the application of sperm sexing technology to semen, as increasing the number of female cattle is essential for expanding the herd while maintaining good genetics. Sexed semen is semen that has undergone a process of sorting and selection to alter the typical proportion of Y-chromosome-bearing sperm (Y-sperm) and X-chromosome-bearing sperm (X-sperm) [4,5]. The sex of newborn calves can be regulated toward a desired sex with over 90% accuracy based on small differences in the content of DNA between the X- and Y-sperm in the cattle, which are 3.7–4.2% dependent on the breed [6]. Primarily, cell sorting by flow cytometry has been employed for sexing sperm. While this technology achieves an accuracy of 85–95% for female calving birth rates, it is associated with high production costs, reduced sperm quality, and diminished fertility [7,8].

Immunological sexing technology offers an alternative method for generating sexed semen. These techniques offer low-cost production and sexed semen of good quality and can easily be applied on a commercial scale. The underlying principle of immunological techniques is based on specific antibodies against the plasma membrane of Y-sperm to separate Y- and X-sperm [9,10]. Currently, numerous studies have successfully generated antibodies specific to Y-sperm, including monoclonal antibodies (Mab) and soluble single-chain fragment variable (scFv) antibodies [[11], [12], [13]]. In a previous study, Mab-1F9 was produced and applied to destroy Y-sperm in conventional semen through the complement system for cytotoxicity reactions, resulting in a high proportion of X-sperm in sexed semen. Compared to conventional semen, sexed sperm from this sexing approach has a high conception rate, with female calves being born at a percentage of 74%. It also has no detrimental impacts on acrosomal integrity or sperm quality [14]. Likewise, the soluble scFv antibody was generated to target male-specific regions on the cell surface of bovine Y-sperm. The scFv antibody was produced from recombinant Escherichia coli TG1 cells incorporating the scFv gene (650 bp), with a high affinity for Y-sperm and slight cross-reactivity with X-sperm [11].

Magnetic-activated cell sorting (MACS) is a novel method that selects dying sperm cells in the primary stages of apoptosis depending on phosphatidylserine externalization; these residues attached to beads and remain in the magnetic field of the column, while live sperm cells are eluted from the column [15]. MACS is a useful technique for selecting cells that are positive for the target antigen. However, successfully purifying various cell types on a large scale without utilizing antibodies specific to the target cell type remains an obstacle [16]. Sringarm et al. (2022) [17] developed MACS by coupling scFv antibodies against Y-sperm with magnetic microbeads. Thus, MACS, which uses magnetic microbeads coupled with scFv antibodies specific to Y-sperm, can be used to sort bull sperm into two fractions: X-enriched and Y-enriched fractions. The benefits of this sexing process include lower costs, greater sexed sperm production, and high-quality post-thaw sperm [14,17]. However, the accuracy (i.e., the sex ratio after in vitro fertilization and the female calf birth rate) of sexed semen produced using this process has not yet been determined.

Acrosomal integrity and vitality are crucial factors when evaluating sperm quality [14]. Flow cytometry is a useful methodology for evaluating and characterizing the physiology and function of cells, with applications for several cell types [18,19]. The assessment of sperm quality using flow cytometry has grown in importance and is increasingly employed for both regular testing and research [20]. Evaluation of the X-/Y-sperm ratio as an indicator of the efficacy of sexing technology can be done using flow cytometry with Hoechst 33342 dye [21]. Moreover, flow cytometry can also be used to assess acrosomal integrity and sperm viability [[18], [19], [20]]. Generally, computer-assisted sperm analysis (CASA) is used to obtain important information on a variety of functional and structural characteristics of sperm related to fertilization capacity [22]. CASA has been used to assess the kinematic characteristics of sperm and describe sperm motility. These factors may have an impact on fertility because they are linked to the hyperactivation traits of post-thaw semen [23]. In vitro fertilization (IVF) is an effective technique for determining sperm fertility [24]. In addition, the pregnancy rates and sex ratio at birth following artificial insemination (AI) must be taken into consideration when evaluating sexed semen [14].

Therefore, the purpose of the present study was to evaluate the efficiency of sperm sexing using MASC coupled with scFv antibodies specific to Y-sperm and the effects on sperm quality parameters, including sperm motility and kinematic characteristics determined using CASA, acrosomal integrity and viability determined using imaging flow cytometry, and the X-/Y-sperm ratio. In addition, the sex ratios resulting from IVF and AI were evaluated in a farm trial.