Open Veterinary Journal, (2026), Vol. 16(4): 2072-2081

Research Article

10.5455/OVJ.2026.v16.i4.12

The effect of toll-like receptor 7/8 activation by ligand (R848) on morphometry, proportion of X-Y chromosome-bearing sperm, and sexed sperm quality of bucks

Nurcholidah Solihati^*, Rangga Setiawan and Siti Darodjah Rasad

Laboratory of Animal Reproduction and Artificial Insemination, Department of Animal Production, Faculty of Animal Husbandry, Padjadjaran University, Sumedang, Indonesia

*Corresponding Author: Nurcholidah Solihati. Laboratory of Animal Reproduction and Artificial Insemination, Department of Animal Production, Faculty of Animal Husbandry, Padjadjaran University, Sumedang, Indonesia.
Email: nurcholidah [at] unpad.ac.id

Submitted: 28/11/2025 Revised: 03/03/2026 Accepted: 17/03/2026 Published: 30/04/2026

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ABSTRACT

Background: Sperm separation is a reproductive biotechnology that separates X- and Y-chromosome-bearing sperm to predetermine offspring sex, offering major benefits for livestock breeding programs. The discovery of an easy, inexpensive, and effective method will be very useful as an alternative to flow cytometry. The swim-up method is a method that meets these criteria. Combining the swim-up method with the addition of a substance to separate X- and Y-chromosome-bearing sperm is very helpful in developing more efficient methods. The activation of TLR 7/8 using the ligand R848 is related to sperm morphometry, the proportion of X and Y chromosome-bearing sperm, and sexed sperm quality.

Aim: This study aimed to determine the effect of activating TLR 7/8 using the ligand R848 on sperm morphometry, the proportion of X- and Y-chromosome-bearing sperm, and sexed sperm quality in bucks.

Methods: Fresh semen samples were collected from 3-year-old bucks using an artificial vagina. A completely random design was used with four treatment levels of ligand (R848) and seven replications. The level treatment of ligand (R848) consisted of T₀=0 µM, T₁=0,3 µM, T₂=0,6 µM, and T₃=0,9 µM. Morphometry, the proportion of X-Y chromosome-bearing sperm, immunostaining, and sexed sperm motility were the evaluated parameters.

Results: Sperm length ranged from 7.02 to 9.99 µm and 8.54 µm on average. Sperm width ranges from 3.08 to 6.01 µm, with an average of 4.61 µm. The sperm head area ranges from 28.02 to 34.14 µm². The proportion of X and Y sperm was significantly different between the upper and bottom layers at treatment 3 (T3), but not for other treatments. The use of ligand (R848) significantly (p < 0.05) affected sperm motility at the bottom layer, but not in the upper layer. The results show that at the bottom layer, the total motility of T3 (79.56%) is significantly lower than that of T0 (89.78%), T1 (87.18%), and T2 (87.01%), whereas T0, T1, and T2 are not significantly different.

Conclusion: Ligand (R848)-mediated activation of Toll-like receptors 7/8 selectively suppresses motility of X-chromosome-bearing sperm in buck, enabling effective enrichment of X-sperm via simple swim-up separation. Morphometric analysis, motility assessment, and immunofluorescence analysis confirmed that the bottom fraction was significantly enriched in functionally impaired X-sperm.

Keywords: Buck, Ligand (R848), Morphometry, Sperm quality, Sperm sexing.

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Introduction

Sperm separation holds vital applications in livestock production, enabling desired sex ratios to improve productivity. Current gold standard techniques, including flow cytometry, rely on DNA content differences between X- and Y-chromosome-bearing sperm but are limited by high costs, operational complexity, and potential sperm damage (Quelhas et al., 2021). It is critical to develop simpler, cheaper methods for exploiting molecular differences between sperm.

The fundamental principle of sperm separation is based on the differences between X- and Y-chromosome-bearing sperm. It has been reported that X-chromosome-bearing sperm have 3%–4% more DNA than Y-chromosome-bearing sperm and have slower motility than Y-chromosome-bearing sperm (Johnson et al., 1989; Garner and Seidel, 2008). The swim-up method relies on sperm motility, which allows highly motile sperm to migrate into the medium’s upper layer against gravity. This procedure helps eliminate some bacteria and debris, which tend to remain in the lower fraction (Henkel and Schill, 2003).

Current sperm separation techniques have been successfully reported. Umehara et al. (2019) used the swim-up method and Toll-like receptors 7 and 8 (TLR7/8) ligand to report that TLR7 and TLR8, membrane-associated receptor proteins, were exclusively expressed at the tail of X-sperm but absent in Y-sperm. TLR7 and TLR8 are exclusively expressed in X-chromosome-bearing sperm (X-sperm), localized mostly to the sperm tail (TLR7) and midpiece (TLR8), suggesting their involvement in the regulation of metabolic pathways in the functional differences between X and Y sperm.

The activation of these receptors by specific ligands, such as Resiquimod (R848), results in a distinct biochemical signaling cascade that selectively suppresses X-sperm motility without affecting Y-sperm function. The activation of TLR7/8 by the ligand (R848) was reported to decrease the motility of X-sperm, thereby allowing for sperm in mice (Umehara et al., 2019). TLR7/8 activation initiates intracellular signaling leading to phosphorylation of glycogen synthase kinase 3 alpha/beta (GSK3α/β). This kinase functions as a key regulator of cellular metabolism.

Zhu et al. (2019) reported that the GSK3α/β protein was highly distributed in the peri-acrosomal, mid-piece, and principal piece of the tail in goat sperm. Phosphorylation of GSK3α/β decreases its activity, which in turn suppresses hexokinase activity, a critical glycolytic enzyme responsible for facilitating glucose conversion to glucose-6-phosphate (Umehara et al., 2019). Hexokinase is primarily localized in the sperm tail and is essential for glycolysis-driven adenosine triphosphate production, which is necessary for flagellar motion. Reduced hexokinase activity decreases glycolytic flux and Adenosine Triphosphate (ATP) generation in X-sperm (Umehara et al., 2025). Inhibition of ATP production, both glycolysis and mitochondrial oxidative phosphorylation contribute to sperm ATP supply. TLR8 activation in the sperm midpiece dampens mitochondrial adenosine triphosphate synthesis. The combined reduction in ATP from glycolysis and mitochondria leads to insufficient cellular energy to sustain normal motility. Metabolic downregulation and motility suppression, as the combined effect of glycolytic and mitochondrial ATP reduction, result in a significant decline in sperm motility parameters, such as velocity and progressive motility. This suppression selectively affects X-bearing sperm without compromising viability or acrosome integrity, rendering it reversible after ligand removal (Umehara et al., 2019).

This biochemical pathway enables a functional sperm-sexing method in which incubation with TLR7/8 agonists selectively restrains X-sperm motility, allowing physical separation by swim-up or density gradient methods. Subsequent immunostaining using TLR7/8 antibodies verifies the enrichment of X- and Y-sperm in the low and high motility fractions, respectively (Setiawan et al., 2024).

The pathway provides a mechanistic basis for improved, cost-effective sperm sexing technologies with minimal cellular damage compared to traditional DNA staining and flow cytometry. It also facilitates the preservation of sperm viability and fertilization potential, which are critical parameters for reproductive applications in both animals and humans (Umehara et al., 2025).

Immunological approaches targeting sperm surface proteins, including TLR7/8 receptors expressed on X-sperm, offer promising alternatives but require further validation (Ren et al., 2021; Hou et al., 2024; Sharma and Sharma, 2024). The knowledge gap lies in integrating immunostaining methods with functional assays to reliably distinguish and separate sexed sperm populations without compromising their fertilizing capacity (Sringarm et al., 2022). Large-scale, longitudinal studies were performed to track fertilization success, pregnancy rates, and offspring development after the use of sexed sperm from different sorting methods (Ren et al., 2021; Lara-Cerrillo et al., 2024). This study aimed to quantify sperm morphometric differences, determine natural X-Y proportions in dairy goats, and evaluate the effect of Resiquimod on sperm sexing efficacy and quality via swim-up separation and immunostaining, with Resiquimod as ligand activation of TLR7/8.

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Materials and Methods

Chemicals and animals

Tris (hydroxymethyl) citric acid, D-glucose, and Resiquimod were used as TLR7/8 ligands, which were purchased from Sigma-Aldrich (St. Louis, CA, USA). Semen was collected from fertile male goats using an artificial vagina. Semen samples were washed by centrifugation at 250 g for 5 minutes with Tris-citrate buffer (pH 7.2) to exclude seminal plasma.

Sperm separation

Semen samples were collected from proven bucks, washed, and incubated with TLR7/8 ligand (R848) at concentrations of 0, 0.3, 0.6, and 0.9 µM in Tris-citrate-glucose buffer at 37°C for 60 minutes. Swim-up separation yielded upper (Y-enriched motile) and bottom (X-enriched less motile) fractions. Sperm head length, width, and area were measured microscopically. Sperm (approximately 300 million) were incubated in 3 ml of Tris-citrate buffer (332 mM Tris, 83 mM citrate, and 22.2 mM glucose, pH 7.2) at 37°C with ligand (R848). In this study, the swim-up test was used based on the method proposed in previous studies (Umehara et al., 2019; Huang et al., 2022). After 60 minutes of incubation, the upper (1 ml) and bottom (1 ml) layers were each transferred to a new tube and then centrifuged at 250 g for 5 minutes. The pellet was suspended in Tris-citrate buffer (Wen et al., 2023).

Semen samples were incubated with the TLR7/8 ligand (R848) at concentrations of 0, 0.3, 0.6, and 0.9 μM in Tris media at 37°C for 60 minutes. This treatment selectively binds TLR7/8 receptors expressed on X-chromosome-bearing spermatozoa. Subsequent swim-up assay separated sperm into upper (highly motile, mainly Y-sperm) and bottom (less motile, mainly X-sperm) fractions. After incubation, swim-up separation allowed the collection of highly motile (upper) and less motile (bottom) sperm fractions, which were hypothesized to be enriched for Y- and X-sperm, respectively.

Morphometric identification

Sperm morphometry from the upper and bottom layers was measured using DP2BSW software (Olympus, IX71). The length, width, and sperm head area (SHA) were measured.

Sperm motility

Sperm motility of the upper and bottom layers was recorded for 5 seconds using video microscopy, consisting of total motility, progressive motility, and non-motile motility.

Immunostaining and TLR7/8 ligand separation

Sexed sperm samples were separated into upper (A) and bottom (B) fractions and subjected to differential interference contrast (DIC), stained with 4’,6-diamidino-2-phenylindole (DAPI) to visualize sperm nuclei consistently across fractions, and immunostaining for TLR7/8 ligands to ascertain the distribution of X- and Y-chromosome-bearing sperm. DIC images confirmed the presence of morphologically intact spermatozoa in both fractions. Nuclear staining by DAPI revealed sperm nuclei as distinct blue fluorescent signals, confirming the presence of sperm cells.

For immunostaining, spermatozoa were incubated with primary antibodies specific to TLR7 and TLR8, which are encoded by genes located on the X chromosome. Following incubation, secondary antibodies conjugated to a green fluorescent dye were used to detect these receptors. Negative controls omitted the primary antibody to ensure staining specificity.

Fluorescent imaging was performed using a confocal microscope, capturing DAPI fluorescence to identify sperm nuclei (blue) and immunofluorescent labeling of TLR7/8 (green) to differentiate X- and Y-sperm populations within each fraction.

Immunostaining with anti-TLR7/8 antibodies and DAPI counterstain confirmed receptor localization and sperm identity (Umehara et al., 2019).

Data analysis

Statistical analysis included analysis of variance and Tukey Hydroxysteroid Dehydrogenase (HSD) post hoc with the StatPlus program, with a significance level of p < 0.05.

Ethical approval

All animal experiments were performed with the approval of the Research Ethics Committee of Padjadjaran University, No: 332/UN6.KEP/EC/2024.

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Results

Morphometric identification and proportion of X-Y chromosome-bearing sperm

Morphometric identification of the length, width, and SHA of dairy goat sperm showed that the sperm head length ranged from 7.02 to 9.99 µm and 8.54 µm on average. Sperm width ranges from 3.08 to 6.01 µm, with an average of 4.61 µm. The SHA ranges from 28.02 to 34.14 µm ². According to this result, sperm have a wide variation in size. This result is in accordance with what has been previously reported, that there is a difference in sperm head size between X-chromosome-bearing sperm (X-sperm) and Y-sperm. X-chromosome-bearing sperm are larger than Y-sperm, which is related to DNA content. The results showed that the average SHA from T0, T1, T2, and treatment 3 (T3) at the upper layer was 31.518, 32.088, 32.09, and 30.39 µm² respectively, and at the bottom layer was 31.81, 32.87, 32.86, and 32.35 µm² respectively. This result suggests that the bottom fraction is larger than the upper fraction.

Analysis of variance showed that the level of the TLR7/8 ligand significantly affected the SHA. This study showed that at T3 with 0.9 µM, the rate of SHA at the bottom fraction (32.347 µm ²) was significantly larger than that of SHA at the upper fraction (Fig. 1). It showed that at the bottom fraction, more X-sperm were found than Y-sperm. Also, it showed that 0.9 µM TLR 7/8 ligand (T3) was effective in separating X-sperm from Y-sperm.

Fig. 1. The effect of TLR 7/8 ligand on the SHA at the upper and bottom layers. T0=Resiquimod (R848) 0 µM; T1=Resiquimod (R848) 0.3 µM; T2=Resiquimod (R848) 0.6 µM; T3=Resiquimod (R848) 0.9 µM. SHA at the bottom layer resulted from T3 with 0.9 µM Resiquimod significantly (p < 0.05) larger than at the upper layer, indicating the presence of X-sperm more than Y-sperm.

Proportion of X-sperm after swim-up and TLR 7/8 ligand treatment

Figure 2 shows the X-sperm proportion at the upper and bottom fractions from each treatment. The natural (control, T0) distribution of X-sperm between the upper and bottom fractions is close to parity following swim-up separation, consistent with the expectation of a near 50:50 ratio in unsorted mammalian semen (Ren et al., 2021; Umehara et al., 2019; Wen et al., 2023). Across treatments with increasing concentrations of Resiquimod (T1: 0.3 µM, T2: 0.6 µM), the proportion of X-sperm in the upper and bottom layers remained statistically similar, indicating minimal separation at lower ligand levels.

Fig. 2. The X-Sperm proportion at the upper and bottom fractions in each treatment. The proportion of X-sperm in the bottom layer at T3 was significantly (p < 0.05) higher than in the upper layer (54.60% vs. 47.46%). This shows that T3 (0.9 µM) is more effective in separating X-sperm than other treatments.

A marked divergence is observed at the highest Resiquimod concentration (T3: 0.9 µM), where the bottom fraction shows a significant increase in X-sperm proportion (indicated by the letter “b”), rising above 54%, compared to the upper fraction that remains below 50% (marked “a”). Statistical grouping (letters above bars) demonstrates that this difference is statistically significant (p < 0.05) at T3, confirming enhanced X-bearing sperm enrichment in the bottom layer.

Effect of TLR7/8 ligand on the sperm motility

Figure 3 shows the effect of the TLR7/8 ligand on the whole parameter of sperm motility. The results of variance analysis showed that the use of TLR 7/8 ligand (R848) had no significant effect on all motility parameters (total motility, progressive motility, and non-motility) at the upper layer. However, it significantly affects (p < 0.05) all motility parameters in the bottom layer. Tukey’s HSD test showed that at the bottom layer, the total motility of T3 (79.56%) was significantly lower than that of T0 (89.78%), T1 (87.18%), and T2 (87.01%), whereas T0, T1, and T2 were not significantly different. These results show that Resiquimod (R848) levels of 0.3 and 0.6 µM have the same percentage of total motile cells as that without the ligand (R848). In contrast, the administration of ligand (R848) at 0.9 µM reduced the total motile level of the bottom layer. The use of TLR 7/8 ligand (R848) had a significant effect on progressive motility, while ligand (R848) at 0.9 µM significantly decreased progressive motility, which was significantly lower than that of T0, but no significant difference between 0.3 and 0.6 µM. The use of TLR 7/8 ligand (R848) also had a significant effect on non-motile sexed sperm, with 0.9 µM significantly higher than T0, T1, and T2.

Fig. 3. The effect of Resiquimod on the sperm motility at upper and bottom layer. The use of Resiquimod (R848) had no significant effect on all parameters of motility at the upper layer. The use of Resiquimod (R848) significantly (p < 0.05) affects total motility, progressive motility, and non-motile in the bottom layer. The total and progressive motilities of T3 bottom layer are significantly (p < 0.05) lower than T3 at the upper layer and T0, but not significant compared with T2 and T1 at the bottom layer. It means that T3 at the bottom layer with 0.9 µM Resiquimod significantly (p < 0.05) decreased the total and progressive motility compared to T3 at the upper layer. The use of Resiquimod (R848) significantly (p<0.05) affected non-motile sperm; the T3 bottom layer with 0.9 µM Resiquimod significantly increased the number of non-motile sperm compared to other treatments. The results indicate that in the bottom layer, there are more X-sperm with lower motility, even non-motile sperm, as a result of the action of the TLR 7/8 ligand.

Immunostaining reveals TLR7/8 expression in the X-sperm enriched fraction

In this study, sperm samples were fractionated into upper (A) and bottom (B) layers and analyzed by DIC, DAPI nuclear staining, and immunostaining targeting TLR7/8 ligands. The DIC images confirmed intact sperm morphology in both fractions, while DAPI staining revealed nuclei in both layers, confirming the presence of sperm.

Distinct immunostaining patterns were observed between the two fractions. The spermatozoa in the bottom fraction exhibited strong green fluorescence, indicating TLR7/8 ligands (Fig. 4B). In contrast, the upper fraction of sperm showed minimal to no immunostaining signal (Fig. 4A). The present immunostaining results support the hypothesis that TLR7 and TLR8 serve as molecular markers for distinguishing X- and Y-sperm. These findings corroborate previous studies demonstrating that TLR7/8 receptors are exclusively expressed on X-sperm, providing a molecular marker for sperm sex differentiation. Activation of these receptors selectively impairs X-sperm motility without compromising viability, enabling efficient separation of X- and Y-bearing sperm based on motility and immunoreactivity. The present data suggest that the bottom fraction is enriched in X-sperm, as indicated by positive TLR7/8 immunostaining, whereas the upper fraction predominantly contains Y-sperm.

Fig. 4. Result of immunostaining of sexed sperm from upper (A) and bottom (B) fractions in three dimensions: (a) original, (b) DAPI, and (c) immunostaining. At the lower layer, sperm emits a green color, indicating the presence of TLR 7/8 ligands. DIC images confirmed the presence of morphologically normal spermatozoa in both upper and bottom fractions (A and B). DAPI staining showed well-defined blue nuclei in both fractions, verifying the presence of sperm cells. Immunofluorescence analysis revealed a clear distinction between sperm populations in the two fractions. The bottom fraction exhibited substantial green fluorescence localized on the sperm surface, confirming the presence of TLR7/8 receptors (B). This finding indicates an enrichment of X-sperm in this fraction, consistent with the genetic localization of TLR7/8 on the X chromosome. In contrast, sperm in the upper fraction showed negligible green fluorescence, suggesting a predominance of Y-chromosome-bearing sperm devoid of TLR7/8 expression (A). The immunostaining results confirm that the bottom sperm fraction is dominated by X-sperm, characterized by the expression of TLR7/8 ligands. This technique holds significant potential for improving sperm sex selection in animal breeding and assisted reproductive technologies. The immunostaining and motility assays establish TLR7/8 ligands as specific biomarkers and functional regulators of X-sperm motility. The described ligand-based separation method enables practical sperm sexing applicable in livestock breeding and assisted reproduction, with high sex selection accuracy and minimal detrimental effects on sperm function.

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Discussion

The impact of the TLR7/8 ligand on the SHA following sperm was evaluated at concentrations of 0, 0.3, 0.6, and 0.9 µM. Sperm samples were incubated with these concentrations and subjected to a swim-up separation method that divides sperm into upper and lower layers based on TLR7/8 ligand-influenced motility characteristics. Statistical analysis of the SHA showed distinct groupings: T3 upper (a), T2 upper (ab), T1 upper (ab), T0 upper (ab), T0 bottom (ab), T1 upper (ab), T2 upper (ab), and T3 bottom (b), where groups labeled with different letters differed significantly (p < 0.05), whereas groups sharing letters did not. Notably, the SHA in T3A was significantly different from that in T3B, indicating a measurable effect of TLR 7/8 ligand concentration on sperm morphology, whereas intermediate treatment groups showed overlapping statistical responses, suggesting subtle or no significant differences.

TLR7/8 ligand exerts its effect by binding to TLR7/8, receptors predominantly expressed on X-bearing sperm, leading to decreased motility and ATP content specifically in this subpopulation. This differential effect permits the enrichment of X-sperm in the bottom layer of the swim-up medium, characterized by relatively larger sperm head dimensions due to higher DNA content compared to Y-sperm, which predominantly remain in the upper layer with smaller head sizes. Although morphometrical comparisons across groups did not always reach statistical significance, a trend toward larger SHA in the lower layer, which was significantly larger than T3 at the upper fraction, corresponds with previous reports asserting that X-sperm exhibit larger head size owing to their increased DNA (around 3.5%–4%) compared to Y-sperm (Umehara et al., 2020).

The mechanisms underlying this selective motility reduction involve decreased ATP production in X-sperm upon TLR7/8 activation by TLR7/8 ligand, impacting flagellar beat and swim capacity (Umehara et al., 2020). These findings are consistent with the decreased progressive motility observed in the lower-layer sperm, which was correlated with increasing TLR 7/8 ligand/Resiquimod concentrations, whereas Y-sperm motility in the upper layer remained largely unaffected. This differential motility allows effective separation during the swim-up procedure, improving the sperm sexing precision without causing significant detriment to sperm morphology.

The study underscores the utility of the TLR7/8 ligand in improving non-flow cytometric sperm approaches by exploiting biochemical and physiological disparities between X- and Y-sperm. Nonetheless, the absence of significant morphometric alterations in the intermediate groups suggests that the optimization of Resiquimod concentration is necessary to maximize enrichment efficiency while maintaining sperm integrity. Further investigations are warranted to assess the fertilization potential and in vivo efficacy of sperm separated using this method to establish practical applications in livestock breeding programs.

The results of the X-Y sperm proportion directly demonstrated the effect of TLR7/8 activation by Resiquimod: X-sperm, which express these receptors, have their motility selectively suppressed through metabolic inhibition, causing them to accumulate in the lower, less motile swim-up fraction (Umehara et al., 2019; Ren et al., 2021). In contrast, Y-sperm lacking TLR7/8 maintain motility and are more likely to reach the upper layer, resulting in a relative depletion of X-sperm in that fraction.

These results validate the use of Resiquimod-mediated TLR7/8 activation as a practical method for sperm separation in livestock. The findings are consistent with recent studies in mice, bovines, and goats that reported targeted X-sperm enrichment via ligand-based metabolic suppression, with no adverse effects on sperm integrity at moderate concentrations (Huang et al., 2022; Wen et al., 2023)

The TLR7/8 ligand Resiquimod (R848) was used to activate TLR7/8 in mouse and bull X-sperm, resulting in decreased glycolytic activity and ATP production, with a consequent reduction in X-sperm motility (Umehara et al., 2019). The use of Resiquimod (R848) in sperm separation using the swim-up method activated TLR7/8 in X spermatozoa. Ren et al. (2021) stated that TLR 7/8 in dairy goats is considered in the spermatozoa tail. It was further reported that activation of TLR 7/8 in X sperm results in inhibition of ATP production from hexokinase activity and mitochondrial activity, which results in reduced energy and motility of X spermatozoa (Umehara et al., 2019; Ren et al., 2021; Wen et al., 2023). This is in line with the results of this study, that is, the presence of Resiquimod (R848) as a ligand of TLR 7/8 reduces the motility of spermatozoa in the bottom layer of the medium, which is thought to contain a lot of X spermatozoa. However, Resiquimod does not affect the motility of spermatozoa in the upper layer, which is thought to contain a large number of Y spermatozoa.

Umehara et al. (2019) reported that ATP production in mitochondria regulates the progressive motility of spermatozoa. This situation causes the progressive motility of spermatozoa in the bottom layer to be lower than that in the upper layer. When ATP production is inhibited, the movement of X spermatozoa tends to be stuck in the bottom layer, and Y spermatozoa will move to the surface of the media because they are not affected by the presence of Resiquimod (R848). Previous research stated that the presence of Resiquimod (R848) did not affect mitochondrial ATP production in Y sperm, so their motility had a normal speed (Ren et al., 2021b).

Zhu et al. (2019) reported that the addition of a GSK3 inhibitor led to an increase in ATP content in goat sperm, suggesting that the GSK3α/β is involved in regulating ATP production in goat sperm. Du Plessis et al. (2015) reported that both glycolysis and oxidative phosphorylation pathways could generate ATP in sperm; the enzymes involved in these pathways are essential for ATP generation. The majority of enzymes involved in the glycolysis pathway are located in the sperm tail and acrosome. Furthermore, Amaral (2022) stated that glycolytic enzymes are primarily localized in the sperm tail, whereas mitochondria accumulate in the midpiece. Zhu et al. (2019) analyzed the activities of Lactate Dehydrogenase (LDH) (an important enzyme for glycolysis), Malate Dehydrogenase (MDH), and Succinate Dehydrogenase (SDH) (two rate-limiting enzymes for oxidative phosphorylation). The addition of the GSK3 inhibitor significantly increased the activities of LDH, MDH, and SDH and induced the acrosome reaction in vitro.

Krawczyk et al. (2010) reported that R848 treatment increased the phosphorylation of GSK3α/β. GSK3α/β influences hexokinase activity and ATP production in dendritic cells following GSK3α/β inhibition. Hexokinase phosphorylation was significantly reduced in sperm in mice, cattle, goats, and rams, suggesting that TLR7 may regulate ATP production via GSK3α. These results suggest that these effects are conserved across mammalian species (Umehara et al., 2020; Ren et al., 2021; Wen et al. (2023).

The use of Resiquimod (R848) in separation only suppresses the motility of X spermatozoa without reducing their ability to fertilize. Nevertheless, it can still be used for artificial insemination after the removal of Resiquimod (R848). The movement of X spermatozoa can be restored by washing the sample. Washing was performed by centrifugation to separate Resiquimod (R848), and the sample was added to the new diluent media. This refers to previous research, which states that the inhibition of spermatozoa motility by Resiquimod (R848) is reversible; the inhibited motility can be mediated by eliminating Resiquimod (R848) in the media (Umehara et al., 2019).

TLR7/8 activation by specific ligands reduces the motility of X-sperm without affecting Y-sperm, facilitating physical separation through differential motility assays. This method offers several advantages over conventional flow cytometry, including reduced cell damage and DNA fluorescent dye obviation. TLR7/8 activation reduces X-sperm motility through intracellular signaling involving glycogen synthase kinase phosphorylation and downstream metabolic inhibition, diminishing ATP production critical for motility. This reversible suppression permits selective separation and retains post-treatment fertilization capability. The proportion of motile sperm in the upper swim-up fraction decreased significantly for X-sperm with TLR7/8 activation, whereas the relative percentage of Y-sperm increased due to unaffected motility (Umehara et al., 2019). TLR7 activation on the sperm tail and TLR8 activation on the midpiece initiate phosphorylation of GSK3α/β, which suppresses hexokinase activity, a key enzyme that converts glucose to glucose-6-phosphate in glycolysis. Reducing hexokinase activity decreases glycolytic ATP production, which is essential for flagellar motility. Additionally, TLR8-mediated mitochondrial inhibition lowers ATP from oxidative phosphorylation. This combined energy deficit selectively impairs X-sperm motility without affecting viability or acrosomal integrity (Umehara et al., 2019).

The reduction in motility caused by TLR7/8 activation is reversible and does not impair sperm viability or fertilization capability, making it a promising tool for sex selection in reproductive technologies (e.g., livestock breeding or assisted reproduction) (Umehara et al., 2019). Sperm swim-up tests combined with TLR7/8 ligand treatment result in slow-moving X-sperm in the lower layers and faster Y-sperm in the upper layers, facilitating their physical separation (Bai et al., 2025). Investigate downstream signaling pathways of TLR7/8 activation in sperm, including hexokinase regulation and mitochondrial function, and assess long-term fertility and offspring outcomes in multiple species after ligand treatment (Umehara et al., 2019; Ren et al., 2021; Pan et al., 2025). TLR7/8 ligand-treated sperm maintain fertilization capacity, enabling embryo production with sex ratio skewing (Umehara et al., 2019; Umehara et al., 2020).

The immunostaining approach described here demonstrates a reliable assay for confirming post-separation sperm sex fractions, which is critical for applications in livestock breeding and human-assisted reproduction where sex selection is desirable. Future work should focus on optimizing antibody specificity and staining protocols for clinical or field use, as well as assessing the reproductive efficacy of sorted X-sperm. Immunostaining revealed a significant difference between the two fractions. In the bottom fraction (B), spermatozoa exhibited significant green fluorescence, indicating TLR7/8 ligands. This fluorescence corresponds to the specific binding of antibodies raised against TLR7/8, receptors encoded by genes located on the X chromosome, thus identifying the sperm as X-bearing. Conversely, the upper fraction (A) showed a negligible immunostaining signal, which is consistent with the predominance of Y-sperm lacking TLR7/8 expression. The green fluorescence observed in the bottom fraction of the immunostaining images corresponds to sperm bound by antibodies targeting TLR7/8, confirming the presence of these receptors and thus the X-sperm fraction (Bai et al., 2025). The absence or minimal green fluorescence in the upper fraction indicates Y-sperm dominance, which lacks TLR7/8 (Umehara et al., 2019).

Research on immunostaining of sexed sperm, TLR7/8 ligands, DIC, DAPI, Fluorescein Isothiocyanate (FITC), sperm morphology, reproductive biology, and sperm fraction analysis has emerged as a critical area of inquiry due to its potential to enhance livestock production efficiency and address sex-linked genetic disorders. The field has evolved from early sperm separation methods based on physical and biochemical differences to advanced immunological and molecular techniques, including flow cytometry and fluorescence-based assays (Sharma and Sharma, 2024). These technologies enable the separation of X- and Y-chromosome-bearing sperm with reported accuracies of 80%–90%, facilitating sex-specific offspring production that benefits the dairy and meat industries by optimizing herd composition and reducing costs (Quelhas et al., 2021).

Immunostaining methods such as DAPI and FITC combined with DIC microscopy provided effective visualization and differentiation of X- and Y-chromosome-bearing sperm, enabling detailed morphometric and viability assessments. Studies have demonstrated that these techniques can identify differences in nuclear size and DNA content, facilitating sex determination in non-sexed and sexed semen samples (Santolaria et al., 2016). Moreover, the application of fluorescence markers, such as DAPI and FITC, combined with DIC microscopy, has improved sperm morphology assessment and viability evaluation (Ling et al., 2022).

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Conclusion

Resiquimod-mediated activation of TLR7/8 selectively suppresses motility of X-chromosome-bearing sperm buck, enabling effective enrichment of X-sperm via simple swim-up separation. Morphometric analysis, motility assessment, and immunofluorescence confirm that the bottom swim-up fraction is significantly enriched in functionally impaired X-sperm. TLR7/8 ligands are also established as specific biomarkers and functional regulators of X-sperm motility. This approach holds significant promise for improving reproductive management and genetic selection in dairy goats and other livestock species.

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Acknowledgments

The authors would like to thank the Ministry of Higher Education, Science and Technology, Republic of Indonesia, as a funder of Fundamental Research Grant 2024.

Conflict of interest

The authors declare no conflicts of interest regarding the publication of this article.

Funding

Funding: This research received funding from the Ministry of Higher Education, Science and Technology, Republic of Indonesia through Fundamental Research Grant 2024, Contract Number 074/E5/PG.02.00.PL/2024.

Authors’ contributions

N.S.: designed the research, was in charge of the experimental design, interpretation, data analysis, and literature study. S.D.R.: literature review, grammar checking. R.S.: research director, experimental design, interpretation, and data analysis, literature review.

Data availability

All data supporting this study’s findings are available within the manuscript. 

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