Serum Mineral Profiles and Testosterone Levels as Integrated Fertility Biomarkers in Boerka Bucks

The Boerka goat is a composite breed resulting from the crossbreeding of Boer goats with Kacang goats. This breed is characterized by predominantly white body coloration, a two-tone coat pattern with a dark brown head, a convex facial profile, horns, pendulous ears, and a slightly concave rump in both males and females. Boerka goats possess desirable growth performance, weight gain, and strong adaptability to humid tropical environments when provided with adequate nutrition (Nugroho et al., 2019). Efforts to enhance reproductive efficiency can be achieved through the application of artificial insemination (AI) technology, whose success largely depends on semen quality (Jha et al., 2020).

Frozen semen plays a critical role in AI programs, as semen collected from genetically superior males can be used to improve the genetic merit of the population. The production of high-quality frozen semen is closely linked to sperm quality. Previous studies have attempted to improve semen characteristics through the administration of PGF2α (Husnurrizal et al., 2021; Panjaitan et al., 2024) and GnRH (Syafruddin et al., 2020; Sutriana et al., 2022). However, these hormonal interventions have limitations, as the enhancement of reproductive performance is temporary and highly dependent on exogenous hormonal stimulation.

Several reports highlight the interaction between nutrition and reproduction in sheep and goats (Vazquez-Armijo et al., 2011; Chao et al., 2023). Among nutritional factors, macro- and microminerals (trace elements) have received particular attention. In cattle, semen that meets the criteria for successful cryopreservation is associated with high serum concentrations of Zn and Fe, suggesting their potential use as biomarkers for freezability (Pipan et al., 2021). To date, no reports are available regarding microminerals as biomarkers of semen quality in goats, despite their potential relevance in predicting post-thaw semen performance.

A variety of accurate methods exist for identifying fertility biomarkers in males; however, many of these methods are time-consuming, expensive, and inefficient because they can only be applied to a limited number of animals at one time (Singh et al., 2018). Therefore, simple, affordable, accurate, and reliable biomarkers are needed to assess semen freezability based on fresh semen or blood analysis. Biomarkers based on macro- and micromineral concentrations (Pipan et al., 2021) and testosterone levels (Hafizuddin et al., 2023) offer promising avenues for application in Boerka goats.

Beyond predicting semen quality, mineral-based biomarkers can also be used to improve semen processing techniques to maintain high post-thaw performance. Optimization may be achieved by supplementing extenders with minerals identified as biomarkers. This supplementation can be performed in the form of mineral nanoparticles (Orzolek et al., 2021). Nanoparticles, defined as particles measuring 1–100 nm, exert effects similar to antibiotics during semen processing by protecting sperm against bacterial contamination and enhancing motility and viability in a dose-dependent manner (Hill and Li, 2017). Previous studies have demonstrated the antioxidant properties of nanoparticles in semen from rams, bulls, rats, and poultry (Singh et al., 2018). The present findings could be further developed for producing mineral nanoparticles identified as biomarkers to support improved semen cryopreservation procedures in Boerka goats. Therefore, this study aimed to identify macro- and micromineral profiles associated with semen quality and freezability in Boerka goats, evaluate the potential of these minerals as biomarkers for predicting post-thaw semen performance, and provide a scientific basis for the future development of mineral-based strategies, including nanoparticle supplementation, to improve frozen semen quality in Boerka goats.

This study was conducted from May to December 2025. The Boerka goats used for semen collection and evaluation were obtained from the UPT for Ruminant Breeding, Lobusona, under the Department of Plantation and Livestock Services of North Sumatra Province. Analyses of macro- and microminerals, including Fe, Zn, Mn, Mg, Ca, and Co, were performed at the Chemical Engineering Laboratory, Faculty of Engineering, Syiah Kuala University, Banda Aceh, while testosterone analysis was carried out at the Physiology Laboratory, Faculty of Veterinary Medicine, Syiah Kuala University.

A total of 10 semen plasma samples and 10 blood serum samples were collected from Boerka bucks (n= 10), all approximately 3 years of age. The animals were maintained at the UPT for Ruminant Breeding, Lobusona, under good management and feeding practices. Semen collection was performed using an electroejaculator. Immediately after collection, semen was evaluated both macroscopically and microscopically. Macroscopic parameters included volume, color, pH, and consistency, while microscopic assessments covered sperm concentration, motility, viability, and abnormalities. Semen samples were classified into fertile (F) and infertile (IF) groups. The fertile group met the criteria of ≥75% motility, ≥60% progressive motility, and ≥75% normal morphology (n= 5). Samples failing to meet at least one of these criteria were categorized as infertile (n= 5).

Blood samples were collected from the jugular vein into EDTA-containing vacutainer tubes, homogenized, and placed in a chilled cool box. Samples were centrifuged at 3000 rpm for 15 minutes, and the resulting plasma was transferred into microtubes and stored at –20°C until analysis.

The concentrations of macro- and microminerals (Fe, Zn, Mn, Mg, Ca, and Co) were measured using spectrophotometry and Atomic Absorption Spectrophotometry (AAS; Shimadzu 700).

Testosterone concentrations were measured on the day of semen collection using an ELISA kit. The testosterone antibody showed 0.8% cross-reactivity with 5α-dihydrotestosterone, 0.9% with androstenedione, 3.3% with 11β-hydroxytestosterone and 19-nortestosterone, and <0.1% with epitestosterone, estrogens, progesterone, cortisol, and danazol. The mean inter-assay coefficient of variation for duplicate samples was 6.71%, and the intra-assay coefficient was 3.28%.

Semen characteristics and macro- and micromineral concentrations in semen plasma and blood serum were presented descriptively. Statistical comparisons between fertile (F) and infertile (IF) groups for fresh semen, post-thaw semen, and mineral concentrations were analyzed using the Wilcoxon test. Correlations between mineral concentrations in blood serum and semen plasma were assessed using Spearman’s correlation coefficient, and adjusted P-values were used to determine statistical significance.

An evaluation of semen characteristics was performed to determine differences in ejaculate quality between fertile and infertile Boerka bucks. The assessment included macroscopic parameters (volume, color, odor, and consistency) and microscopic parameters (sperm concentration, mass motility, individual motility, viability, and morphological abnormalities). These parameters were used to assess the physiological capability of spermatozoa to support the fertilization process. Overall, the findings revealed clear variations in semen quality between the two groups. The fertile Boerka bucks exhibited higher mean semen volume and sperm concentration compared with the infertile bucks. Individual motility and sperm abnormalities also differed markedly between groups, whereas mass motility and viability were relatively similar (Table 1).

The evaluation of semen characteristics revealed variations between fertile and infertile Boerka goats in both macroscopic and microscopic parameters. Overall, the semen volume of fertile Boerka goats (1.66±0.90 mL) was higher than that of infertile goats (1.45±1.20 mL) (Table 1). This difference indicates that the secretory activity of the accessory glands is more optimal in fertile males, contributing to an increased ejaculate volume. The uniform color and odor of semen in both groups (creamy and typical) indicate no abnormalities in the composition of seminal plasma, while the thick consistency suggests relatively high sperm concentrations in both groups.

Microscopically, the sperm concentration of fertile goats (208.20±142.07 × 10⁶/mL) was significantly (P < 0.05) higher than that of infertile goats (180.20±103.05 × 10⁶/mL) (Table 1). This elevated concentration reflects more efficient spermatogenesis, particularly in the proliferation and differentiation stages of spermatids into mature spermatozoa. This process is strongly influenced by intratesticular testosterone levels, Sertoli cell activity, and adequate testicular blood perfusion (Hafez and Hafez, 2016). Meanwhile, individual motility in fertile goats (80.52±0.85%) was also higher than in infertile goats (72.18±17.05%), indicating that spermatozoa from fertile animals possess better progressive motility. Sperm motility is a key parameter influenced by molecular factors such as proAKAP4 and supplementation treatments such as EGF. Good motility is essential for supporting successful fertilization (Ribas-Maynou et al., 2023).

In contrast to these parameters, mass motility and viability did not show clear differences between the groups (Table 1). The relatively similar values of mass motility (2.40±0.55 vs. 2.50±0.76) indicate that the collective movement of spermatozoa in both groups remains within the normal range, while comparable viability (66.91±16.96% vs. 68.75±13.54%) suggests that membrane integrity is still well-maintained in both groups. Nevertheless, morphological abnormalities were lower in the fertile group (2.22±1.49) compared with the infertile group (12.65±5.92).

Reproductive hormones exert differential impacts on semen quality. Elevated LH and FSH levels tend to reduce sperm parameters, while testosterone plays a supportive but not strongly dominant role. Thus, endocrine evaluation combined with semen analysis is essential in assessing infertility (Long et al., 2025). Therefore, the higher concentration and individual motility observed in the fertile group are likely related to more stable reproductive hormone levels and a better physiological response of testicular tissues. This finding aligns with previous studies reporting that males with higher sperm motility and membrane integrity have higher conception success rates (Jalius and Depison, 2008). Consequently, superior semen characteristics in fertile Boerka goats may serve as physiological indicators in selecting high-quality breeding males.

However, this study has several limitations, including a limited sample size and the absence of hormonal profiling and pregnancy tests to confirm the direct association between semen quality and fertilization ability. Further studies integrating semen quality parameters with testosterone levels, sperm DNA integrity, and conception rates are required to obtain a more comprehensive understanding of the factors determining fertility in Boerka bucks.

The results showed that testosterone levels in fertile Boerka goats (25.9±9.23 ng/mL) were significantly (P < 0.05) higher than in infertile goats (15.5±7.83 ng/mL), as presented in Table 1. The testosterone concentration in fertile Boerka goats is relatively high compared to other goat breeds. In Gembrong goats, testosterone levels were reported at 13.30±3.90 ng/mL. In Kacang goats, the mean (± SD) testosterone concentrations in adult males and females were 7.25±1.45 ng/mL and 0.95±0.77 ng/mL, respectively (Gholib et al., 2016).

Table 1: Semen characteristics and testosterone levels of fertile and infertile Boerka bucks.

Correlation analysis demonstrated that testosterone levels were positively associated with all semen quality parameters in Boerka goats, although the strength of the correlations varied across variables. Testosterone showed a moderate positive correlation with sperm concentration (r = 0.42) and a strong, significant correlation with motility (r = 0.66; P < 0.05) and sperm viability (r = 0.70; P < 0.05), as presented in Table 2. These findings confirm that increases in testosterone levels tend to be followed by improvements in semen performance, particularly sperm motility and survival, which are key indicators of male fertility.

Table 2: Correlation between testosterone concentration and semen quality in Boerka goats.

The significant differences observed between the fertile and infertile groups indicate that fertility status is strongly associated with testosterone concentration. These findings are consistent with the report of Bodu et al. (2025), who stated that fertility in male livestock is influenced by testosterone as a biomarker, although it remains interconnected with other reproductive parameters. Testosterone is produced by Leydig cells in response to luteinizing hormone (LH) secretion and plays a critical role in the development of male characteristics. It has been reported that men and male dogs with poor semen quality exhibit lower testosterone concentrations. In circulation, testosterone is largely bound to proteins primarily albumin and, more specifically, sex hormone-binding globulin (SHBG) while only the free fraction exerts biological activity (Laurent, 2016; Almeida et al., 2017; Holst and Nilsson, 2023).

Testosterone plays an essential role in maintaining spermatogenesis, the function of accessory reproductive glands, and libido (Rojas-Zambrano et al., 2025). Therefore, the lower testosterone levels in the infertile group indicate impaired testicular function or endocrine regulation, which subsequently affects semen quality and quantity. The significant differences between the fertile and infertile bucks further underscore the central role of testosterone in supporting the reproductive function of Boerka goats.

These results align with the findings of Baharun et al. (2022), Rajak et al. (2014), and Dasrul et al. (2020). Testosterone concentration shows a positive correlation (P < 0.01) with semen characteristics such as sperm motility (0.813), normal morphology (0.639), and libido (0.952) (Baharun et al., 2022). Rajak et al. (2014) and Dasrul et al. (2020) also reported a relationship between testosterone levels and semen characteristics particularly motility and sperm morphology in crossbred bulls (Friesian Holstein × Tharparkar) and Aceh cattle. Similarly, Gulia et al. (2010) reported that testosterone deficiency can reduce semen quality.

The findings of this study support the potential use of testosterone concentration as a fertility biomarker in Boerka bucks. Physiologically, the strong positive correlation between testosterone and both motility and viability indicates that this androgenic hormone is a key determinant of semen quality. Motility and sperm morphology are considered the most important factors in determining semen quality, as only motile spermatozoa are capable of fertilizing the oocyte (Morrell and Rodriguez-Martinez, 2009). In addition to motility, the ability of spermatozoa to fertilize the oocyte depends on normal morphology and intact chromatin (Nagy et al., 2013).

In this study, motility and viability showed a significant correlation with testosterone (P < 0.05), whereas sperm concentration exhibited a moderate but non-significant correlation with testosterone (P > 0.05). This finding is consistent with the report of Alba et al. (2024), which states that testosterone enhances membrane stability, acrosomal integrity, and motility capacity through the modulation of key proteins in the sperm tail, collectively influencing sperm motility and morphology. Testosterone affects the expression of oxidative enzymes, antioxidants, and structural proteins that maintain motility and reduce oxidative damage. The moderate and non-significant correlation between testosterone and sperm concentration suggests that other factors such as ejaculation frequency, nutrition, or physiological status may influence sperm numbers.

These findings provide practical implications, indicating that testosterone measurement may serve as a fertility biomarker for selecting Boerka bucks for semen freezing programs. However, testosterone alone cannot serve as the sole indicator of fertility, as several semen parameters are influenced by management, health, age, and environmental factors. Therefore, combining testosterone evaluation with semen quality analysis is necessary for more accurate reproductive assessments. Overall, the significant correlations between testosterone, motility, and viability underscore the central role of this hormone in supporting the reproductive performance of Boerka bucks. These conclusions are consistent with the literature, which emphasizes that the stability of androgenic hormones is crucial for successful spermatogenesis and male fertility.

Minerals are essential components for maintaining physiological balance and reproductive function in males. The availability of macro- and microminerals in the blood strongly influences spermatogenesis, semen quality, and the fertilizing capacity of spermatozoa. Minerals such as calcium (Ca), phosphorus (P), zinc (Zn), iron (Fe), and magnesium (Mg) play important roles in enzymatic regulation, membrane stability, and mitochondrial activity in testicular and sperm cells. Imbalances whether deficiencies or excess can impair sperm quality and ultimately lead to infertility (Fallah et al., 2018).

To determine the relationship between mineral status and male fertility, serum macro- and micromineral levels were measured in fertile and infertile Boerka bucks on the day of semen collection. The results of the mineral concentration analysis are presented in Table 3.

The analysis revealed significant (P < 0.05) differences in the concentrations of several macro- and microminerals between fertile and infertile Boerka bucks. Levels of calcium (Ca), iron (Fe), zinc (Zn), and phosphate (P) were significantly higher in fertile bucks than in infertile bucks (P < 0.05), whereas cobalt (Co), magnesium (Mg), and manganese (Mo) did not differ markedly between the groups (Table 3).

Table 3: Concentrations of macro- and microminerals in blood serum on the day of semen collection.

The higher Ca concentration in fertile bucks indicates its role in supporting spermatogenesis and sperm motility. Calcium functions as a key regulator of sperm physiology, playing essential roles in capacitation, hyperactivation, the acrosome reaction, and fertilization (Garriga et al., 2026). Reduced Ca levels in infertile bucks may reflect impaired ionic metabolism affecting germ cell function. In males, calcium deficiency disrupts the regulation of sperm motility and capacitation, both of which are critical for successful fertilization (Yahyavi et al., 2024). However, Ca exhibited a negative correlation with all semen parameters, particularly sperm viability (r= −0.474; P= 0.420). This suggests that excessively high Ca concentrations do not necessarily improve semen quality. Ca²⁺ supplementation increases metabolic activity but may also trigger capacitation-like changes that impair sperm functionality. Intracellular Ca²⁺ accumulation may reduce motility and disrupt metabolic balance, potentially due to H⁺ ion accumulation in the mitochondrial intermembrane space (Garriga et al., 2026).

The higher Fe concentration in fertile bucks highlights its role in enzymatic systems and testicular cellular respiration. Fe showed a moderate positive correlation with sperm motility (r= 0.495; P= 0.096), indicating that increased serum Fe tends to enhance sperm motility (Table 4). This aligns with Fe’s involvement in mitochondrial oxidative

Table 4: Correlation analysis between semen quality and blood concentrations of Ca, Fe, Zn, and P in Boerka bucks.

processes that generate energy for sperm movement. Fe also acts as an antioxidant, serving as a cofactor for catalase, which protects spermatozoa from oxidative damage. However, excessive Fe may induce lipid peroxidation and membrane damage, negatively affecting motility. Overall, higher Fe levels are associated with improved sperm motility and fertility potential (Rodriguez et al., 2021). Iron is an essential component of cytochrome and catalase, contributing to energy metabolism in Sertoli and Leydig cells. Iron deficiency increases oxidative stress, reduces antioxidant enzyme activity, and ultimately impairs reproductive function (Tsao et al., 2022).

Zinc concentration in semen is strongly associated with semen quality parameters, and Zn deficiency is a major risk factor for reduced semen quality (Osadchuk et al., 2021). This finding is consistent with the present study, in which fertile bucks displayed significantly higher Zn levels than infertile bucks (P < 0.05; Table 3). Zn showed positive correlations with sperm concentration (r = 0.196; P = 0.042) and viability (r = 0.390; P = 0.046), as shown in Table 4. Numerous studies confirm that zinc ions (Zn²⁺) play a major role in male fertility. Zn influences multiple sperm functions, including motility, capacitation, and acrosomal exocytosis processes essential for fertilization. Across vertebrate and invertebrate species, Zn²⁺ modulates sperm motility. In humans, high seminal Zn²⁺ concentrations reduce motility, whereas lowering Zn²⁺ enhances motility. During epididymal transit, reduced intracellular Zn²⁺ enables the development of progressive and hyperactivated motility during capacitation. Mechanistically, Zn²⁺signals through the zinc-sensing receptor GPR39 located in the sperm tail and acrosome. Activation of GPR39 triggers intracellular cascades involving increased cAMP, activation of CatSper Ca²⁺ channels, elevated intracellular Ca²⁺, PKA activation, and the Src–EGFR–PLC pathway. These pathways induce hyperactivated motility and regulate F-actin remodeling required for capacitation and acrosomal exocytosis (Allouche-Fitoussi and Breitbart, 2020). Conversely, Zn deficiency impairs spermatogenesis, increases sperm abnormalities, and reduces serum testosterone levels (Fallah et al., 2018).

Similarly, higher P concentrations in fertile bucks support the energetic metabolism of sperm, particularly ATP production, which is essential for motility and viability (Yousef et al., 2017). Suwimonteerabutr et al. (2023) reported that supplementing semen extenders with 0.04% phosphorus and vitamin B12 improved sperm motility, kinetic parameters, and overall sperm quality. In the present study, phosphorus showed a positive but nonsignificant correlation with semen quality, particularly sperm concentration (r= 0.226; P= 0.774), as presented in Table 4. Phosphate plays a key role in sperm energy metabolism, especially in ATP formation required for motility and the acrosome reaction (Yousef et al., 2017). Although nonsignificant, this positive trend suggests a potential contribution of phosphorus to efficient sperm function.

In general, these results reinforce the view that blood mineral status particularly Ca, Fe, Zn, and P can serve as physiological indicators for assessing the reproductive status of male animals. Deficiencies in these minerals have the potential to disrupt spermatogenesis and reduce fertility performance (Yimer et al., 2023). Conversely, the absence of significant differences in Mg and Co levels indicates that these two minerals remain relatively stable in the basal metabolism of goats, with no evident functional differences related to fertility under these physiological conditions. Correlation analysis was conducted to determine the relationship between macro-minerals (Ca and P) and micro-minerals (Fe and Zn) in blood serum with semen quality characteristics of Boerka goats, including sperm concentration, motility, and viability. These minerals were selected because they are known to exert significant influences distinguishing fertile and infertile males.

This study demonstrates that semen quality in Boerka bucks is closely linked to their mineral and hormonal profiles. Fertile males exhibited superior semen characteristics and significantly higher serum testosterone, along with elevated concentrations of Ca, Fe, Zn, and P. Correlation analyses showed that Fe was positively associated with sperm motility, while Zn correlated with sperm concentration and viability. Testosterone exhibited strong positive correlations with all semen parameters. These findings indicate that serum Fe, Zn, and testosterone are key physiological indicators of fertility in Boerka bucks. Mineral–hormonal profiling thus offers a promising strategy for identifying fertile males and improving the efficiency of breeding and artificial insemination programs.

The author expresses gratitude to the Rector of Universitas Syiah Kuala for funding support through the Penelitian Professor for the fiscal year 2025, with contract number 347/UN11.L1/PG.01.03/14138-PTNBH/2025.

This study provides the first evidence that integrated serum mineral–hormonal profiling, particularly involving iron, zinc, and testosterone levels, can reliably differentiate fertile and infertile Boerka bucks. By demonstrating strong mineral–semen quality relationships in this composite meat breed, the research introduces Fe and Zn as potential physiological biomarkers for buck fertility, offering a novel diagnostic approach to enhance breeding selection and artificial insemination efficiency.

Conceptualization: TNS, SW and HH. Methodology, formal analysis, and investigation: EMS, TA, and HH. Writing original draft, review and editing: TNS, HH, EMS, and SW. All authors have read and agreed to the published version of the manuscript.

Ethical approval was not required for this study based on the recommendation of the Ethical Feasibility Team of the Faculty of Veterinary Medicine, Universitas Syiah Kuala, as no experimental or invasive procedures were applied to the animals and semen collection was conducted using a routine and standardized procedure.

The authors declare that generative AI and AI-assisted technologies were used solely to support the development of the manuscript framework and to improve the clarity, grammar, and academic quality of the English language. No AI tools were used for data analysis, data interpretation, figure generation, or drawing scientific conclusions. All scientific content, analyses, and interpretations remain the sole responsibility of the authors.

The authors have declared no conflict of interest.