JAHP_13_4_1252-1260

Introduction

Bali bulls (Bos indicus) are an indigenous breed with considerable economic and cultural significance in Indonesia, valued for their adaptability to local environmental conditions and genetic resilience. However, despite their potential, reproductive performance in Bali bulls is often constrained by nutritional deficiencies and suboptimal management, which can limit semen quality, libido, and hormonal balance. These limitations ultimately affect the efficiency of breeding programs and genetic improvement efforts. Addressing these challenges is crucial for supporting the national goals outlined in Asta Cita Indonesia, which seeks to enhance livestock productivity and food security through sustainable development of native breeds.

Nutrition plays a fundamental role in regulating reproductive functions in bulls. Deficiencies in essential nutrients, antioxidants, and bioactive compounds can impair endocrine activity, reduce semen quality, and lower sexual behavior (Byrne et al., 2023; Zeng et al., 2023). Supplementation with natural plant-based additives has gained attention as an effective, environmentally friendly strategy to overcome these nutritional gaps. Specifically, leaf powders from Moringa oleifera, Gliricidia sepium, and Indigofera zollingeriana are rich in vitamins, minerals, antioxidants, and phytochemicals such as flavonoids and phenolic compounds, which have been shown to positively influence reproductive physiology (Jamili et al., 2021; Antari et al., 2023).

Previous studies have demonstrated the benefits of phytogenic leaf supplements across various livestock species. For example, Moringa supplementation has been associated with increased testosterone secretion and improved semen volume in bulls (Ismail et al., 2025). Gliricidia has been shown to enhance reproductive hormone concentrations and antioxidant capacity in goats (Assan et al., 2025), while Indigofera supplementation improved libido and semen quality in sheep (Somanjaya et al., 2022). Despite these encouraging results, comparative studies on the effects of these leaf powders in Bali bulls remain limited.

Most research on phytogenic feed additives in ruminants has primarily focused on growth performance, feed efficiency, or general health parameters, with very few studies investigating their role in reproductive performance. This gap is particularly evident in Bali bulls, a native Indonesian breed valued for its adaptability but constrained by relatively low reproductive efficiency. To address this knowledge gap, the present study was designed to evaluate the effects of dietary supplementation with Moringa, Gliricidia, and Indigofera leaf powders on key reproductive indicators, including libido, semen quality (volume and pH), and hormonal levels (testosterone and adiponectin).

The findings of this study are expected to provide scientific evidence supporting the potential of these natural feed supplements as practical interventions to enhance reproductive performance and contribute to the genetic improvement of Bali bulls, an important indigenous breed for sustainable livestock development in Indonesia.

MATHERIALS AND METHODS

Experimental design

This research was conducted at the Laboratory of Animal Reproduction, Faculty of Animal Science, Hasanuddin University, Indonesia, over a period of 90 days. The experimental subjects were 15 Bali bulls, divided into three treatment groups based on feed supplementation using leaf powders. The basal ration provided to all bulls met the nutritional standards recommended for breeding bulls to ensure adequate maintenance and reproductive performance throughout the study. The treatment groups included:

T1: Supplementation with Moringa leaf powder
T2: Supplementation with Gliricidia leaf powder
T3: Supplementation with Indigofera leaf powder

Each group consisted of five cattle that received supplementation at consistent doses and frequencies throughout the experimental period. The experimental diets were formulated on a dry matter (DM) basis and consisted of phytogenic leaf powder according to treatment (10%), tofu waste (10%), molasses (4%), premix (0.5%), salt (0.5%), rice bran (5%), and elephant grass (70%). The rations were designed to be isoenergetic and isoproteic, with nutrient composition adjusted based on the type of leaf powder used. Specifically, the formulated ration for T1 (Moringa leaf powder) contained 13.41% crude protein (CP) and 61.38% total digestible nutrients (TDN), T2 (Gliricidia leaf powder) contained 13.22% CP and 62.00% TDN, and T3 (Indigofera leaf powder) contained 13.98% CP and 63.00% TDN.

The Moringa, Gliricidia, and Indigofera leaves were air-dried under shade at room temperature, ground using a mechanical grinder, and stored in airtight containers until use. Each treatment diet was formulated to meet the requirements for iso-protein and iso-energy according to SNI 3148-2:2017 (Indonesian National Standard, 2017) for breeding bulls, which specifies maximum moisture (14.00%), maximum ash (12.00%), minimum crude protein (12.00%), maximum crude fat (6.00%), calcium (0.60–0.80%), phosphorus (0.30–0.60%), maximum aNDF (35.00%), minimum UDP (4.80%), maximum total aflatoxins (200 μg/kg), and minimum TDN (65%).

All bulls were housed in individual pens with free access to clean water, natural ventilation, and daily health monitoring by trained staff. Management practices followed institutional animal care guidelines. The experimental design used was a completely randomized design (CRD) with three replications. All procedures involving animals were approved by the Animal Ethics Committee of the Faculty of Animal Husbandry, Hasanuddin University (Approval No. 11/UN412/EC/V2025).

Semen measurement

Semen samples were collected from Bali bulls using the artificial vagina (AV) method, with the AV prepared by filling it with water heated to 42–45°C (Diansyah et al., 2024). Immediately after collection, semen volume was measured directly using a graduated collection tube attached to the AV, and semen pH was measured using pH indicator paper.

Hormone assay

Blood samples were collected from the jugular vein of each Bali bull using sterile 10 mL syringes and transferred into vacutainer tubes without anticoagulants (Alfian et al., 2025). Samples were allowed to clot at room temperature for 30 minutes before centrifugation at 3000 rpm for 15 minutes to separate the serum. The serum was then carefully pipetted into sterile microtubes and stored at –20°C until analysis (Ferrer et al., 2022). Serum testosterone and adiponectin concentrations were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (Signalway Antibody, #EK0019). The assays were performed according to the manufacturer’s instructions. Briefly, samples and standards were added to wells pre-coated with specific antibodies and incubated for the recommended time at room temperature. After washing to remove unbound substances, enzyme-conjugated secondary antibodies were added, followed by substrate solution to produce a measurable color change. The reaction was stopped after the specified time, and absorbance was measured using a microplate reader at 450 nm (Baharun et al., 2021).

Data analysis

Data were tested for normality using the Shapiro-Wilk test and homogeneity of variance using Levene’s test. Parameters that met these assumptions were analyzed by one-way analysis of variance (ANOVA) to assess the effect of feed supplementation, followed by Tukey’s Honestly Significant Difference (HSD) test for multiple comparisons among treatment groups. Pearson correlation analysis was performed to evaluate relationships among hormone levels, libido, semen volume, and semen pH. Statistical significance was set at p < 0.05, and all analyses were conducted using SPSS version 27.

RESULTS

Effect of feed supplementation on libido in Bali bulls

The results showed a significant effect of supplementation on libido in Bali bulls (Figure 1). At the initial measurement, libido scores among T1, T2, and T3 groups were similar (around 1.38–1.42) with no significant differences (p > 0.05). Over the course of the experimental period, libido scores increased in all groups, reaching values above 2.0 by the final measurement. Although the differences between groups at later times were not consistently significant, T1 tended to have higher libido scores than T2 and T3 at day 30 (1.80±0.45 vs. 1.60±0.89 and 2.00±0.71, respectively), with overlapping significance letters indicating partial significance (p < 0.05). By days 60 and 90, libido scores among the groups converged (approximately 2.2–2.4), showing no significant difference.

Effect of feed supplementation on semen volume and ph in Bali bulls

Semen volume showed a gradual increase in all treatment groups throughout the experimental period (Figure 2), with initial measurements relatively similar across groups (T1: 3.1 ± 0.74 mL, T2: 2.96 ± 1.42 mL, T3: 3.2 ± 0.84 mL). By the final measurement, volumes increased to 3.56 ± 0.54 mL (T1), 3.6 ± 0.55 mL (T2), and 3.5 ± 0.46 mL (T3), although no significant differences were observed between treatments at any time point (p > 0.05). Meanwhile, semen pH values across all groups remained within the normal physiological range for Bali bulls, approximately between 6.28 and 6.84 (Figure 3). Minor fluctuations occurred during the study but were not statistically significant (p > 0.05). Group T1 showed a slight increase in pH from 6.48 ± 0.52 to 6.84 ± 0.22, T2 exhibited a slight decrease at midpoints before stabilizing, and T3 maintained relatively consistent pH values throughout.

Effect of feed supplementation on hormone Levels in Bali bulls

Testosterone levels increased significantly over time in all treatment groups (Figure 4). At day 0, testosterone levels were similar among groups (T1: 2.95 ± 0.2 ng/mL, T2: 3.48 ± 0.4 ng/mL, T3: 3.09 ± 0.19 ng/mL), with no significant differences (p > 0.05). By day 90, testosterone levels rose markedly, reaching 4.7 ± 0.14 ng/mL in T1, 5.39 ± 0.17 ng/mL in T2, and 4.87 ± 0.14 ng/mL in T3, with significant differences observed between groups at intermediate time points (p < 0.05). The T2 group consistently exhibited the highest testosterone levels throughout the study. Similarly, adiponectin levels showed a gradual increase across all groups (Figure 4). Initial adiponectin levels were lowest in T3 (12.66 ± 0.24 µg/mL) compared to T1 (13.94 ± 0.77 µg/mL) and T2 (13.33 ± 0.05 µg/mL). By day 90, adiponectin levels increased significantly to 14.58 ± 0.22 µg/mL (T1), 14.39 ± 0.13 µg/mL (T2), and 13.81 ± 0.12 µg/mL (T3) (p < 0.05).

Correlation analysis on Bali bulls during the experimental period

Pearson correlation analysis revealed several notable relationships among the measured parameters in Bali bulls (Figure 5). Testosterone levels showed a significant positive correlation with adiponectin levels (r = 0.455, p < 0.01), indicating a strong association between these two hormones. Additionally, testosterone was positively correlated with libido (r = 0.312, p < 0.05), suggesting that higher testosterone levels are associated with increased sexual behavior. Semen volume exhibited a moderate positive correlation with testosterone (r = 0.247), although this was not statistically significant. Libido and semen volume did not show a significant correlation (r = -0.130, p > 0.05), nor did semen pH correlate significantly with any other parameters.

DISCUSSION

This study provides suggestive evidence that dietary supplementation with Moringa oleifera, Gliricidia sepium, and Indigofera zollingeriana leaf powders significantly improved reproductive parameters in Bali bulls, including libido, semen quality, and hormone profiles. All three supplementation treatments effectively enhanced libido, with each group showing a significant increase over the experimental period. While the Gliricidia group demonstrated the highest libido scores, it is important to note that this group also exhibited elevated baseline values at day 0, suggesting that initial physiological status partially influenced the observed results. Nonetheless, the upward trends across all groups indicate that each leaf powder positively modulated sexual behavior, likely through their bioactive compounds impacting the hypothalamic-pituitary-gonadal axis and stimulating testosterone production (Syarifuddin et al., 2022; Zhao et al., 2024).

The supplementation period of 90 days was critical to allow sufficient time for the bioactive compounds in the leaf powders to exert their physiological effects on the reproductive system of Bali bulls. Spermatogenesis in bulls typically requires approximately 61 days to complete, and changes in hormone levels and semen quality often manifest only after sustained nutritional intervention (Cojkic and Morrell, 2023). Therefore, the gradual increases observed in libido, testosterone, adiponectin, and semen volume from day 0 to day 90 likely reflect cumulative improvements resulting from continuous exposure to antioxidants, vitamins, and phytochemicals present in the supplements. This time-dependent effect emphasizes the importance of consistent, long-term supplementation in modulating reproductive performance and achieving measurable benefits.

Although this study did not include a negative control group, the use of three different phytogenic leaf powders as comparative treatments provided a practical and scientifically valid framework to assess their relative efficacy. Because all animals received the same basal ration formulated according to breeding bull nutritional standards, any observed differences can reasonably be attributed to the phytogenic supplementation itself, thereby ensuring meaningful and reliable comparisons.

The absence of a negative control does not necessarily weaken the study design, as comparative approaches are widely adopted in nutritional research. A systematic review of nutrition education interventions reported that about one-third of studies used inactive controls, which are considered weaker designs, while nearly two-thirds employed active controls, which are regarded as stronger (Byrd-Bredbenner et al., 2017). Similarly, various designs in nutrition clinical trials, such as quasi-experimental and multi-arm randomized studies, often compare active treatments rather than including a negative control (Mirmiran et al., 2023). This is further supported by the application of N-of-1 trials, which typically evaluate baseline values or alternate treatments instead of relying on a negative control (Potter et al., 2021).

In this study, improvements in reproductive indicators were observed following supplementation with Moringa, Gliricidia, and Indigofera leaf powders. These effects may be related to the phytochemical composition of the supplemented leaves, which are known to contain antioxidants, flavonoids, and phytoestrogen-like compounds. Supporting this interpretation, previous studies have demonstrated that Moringa oleifera seeds and leaf extracts significantly reduce oxidative stress markers such as malondialdehyde (MDA) while increasing antioxidant enzymes like glutathione S-transferase (GST) and superoxide dismutase (SOD) in testicular tissues (Hamed et al., 2024).

Although fewer studies are available for Indigofera and Gliricidia, their antioxidant activities have also been linked to reduced oxidative stress and enhanced Leydig cell function, potentially contributing to testosterone secretion (Monageng et al., 2023). The beneficial effects of Moringa, in particular, may also be attributed to its rich content of vitamins A, C, and E, essential amino acids, and high levels of flavonoids and phenolic compounds, which enhance antioxidant defense and promote steroidogenesis (Mohlala et al., 2023; Gad et al., 2024). Collectively, these antioxidant and phytochemical properties can help preserve testicular function, support testosterone synthesis, and thereby contribute to improvements in libido and semen quality.

Gliricidia sepium leaves contain significant amounts of tannins, saponins, and flavonoids, compounds known for their antioxidant and anti-inflammatory properties (Wafaey et al., 2023). These bioactives may protect reproductive tissues from oxidative damage and support endocrine function, which aligns with the higher testosterone levels observed in the Gliricidia group. The presence of saponins is also notable for its potential role in enhancing steroid hormone production by stimulating cholesterol uptake in Leydig cells (Raju et al., 2021).

Indigofera zollingeriana leaves are rich in alkaloids, flavonoids, and phenolic acids, which possess antioxidant and immunomodulatory effects (Rusli et al., 222). These compounds may contribute to the observed improvements in libido and semen parameters by supporting cellular metabolism and protecting spermatozoa from oxidative damage, enhancing overall reproductive health.

In this study, semen volume showed a progressive increase across all groups during the experimental period, although the differences were not statistically significant (p > 0.05). These results are in line with previous reports linking improved nutritional and antioxidant status to enhanced seminal plasma production and sperm viability (Vigolo et al., 2022; Ferrer et al., 2024). The absence of clear intergroup differences suggests that the observed response may have been driven more by general nutritional support from supplementation rather than by the specific phytochemical composition of each leaf powder.

Semen pH remained within physiological norms throughout the study, indicating that supplementation did not disrupt seminal fluid homeostasis, which is crucial for preserving sperm motility and fertilizing capacity (Wang et al., 2021). Taken together, the increases in semen volume and the stability of seminal pH should be regarded as indicative trends rather than definitive effects, warranting further confirmation in studies with larger sample sizes.

Significantly elevated testosterone and adiponectin levels post-supplementation highlight the hormone-modulating effects of these phytogenic additives. The Gliricidia group’s superior testosterone levels may be attributable to its potent antioxidant constituents that protect Leydig cell function and facilitate steroidogenesis (Monageng et al., 2023). Adiponectin’s increase suggests enhanced metabolic homeostasis, with implications for energy regulation and reproductive capacity (Choubey et al., 2021). The strong positive correlations between testosterone and adiponectin, as well as testosterone and libido, emphasize the integrated hormonal regulation essential for reproductive competence (Su et al., 2023). The observed correlation between testosterone and libido (r= 0.312) was weak, and thus should only be considered as a preliminary indication of association rather than strong evidence.

The significant positive correlation observed between testosterone and adiponectin levels suggests an intricate interplay between metabolic and reproductive hormones in Bali bulls. Testosterone, a key androgen hormone produced by Leydig cells, directly regulates spermatogenesis and libido by modulating gene expression in reproductive tissues (Lei et al., 2025). Adiponectin, primarily known for its role in energy homeostasis and insulin sensitivity, also influences reproductive function by acting on the hypothalamus and gonads to regulate hormone secretion and steroidogenesis (Li et al., 2024; Kurowska et al., 2022). Elevated adiponectin levels may enhance Leydig cell responsiveness to luteinizing hormone, thereby promoting testosterone synthesis and supporting sexual behavior, which explains the positive correlation between these two hormones. The positive correlation between testosterone and libido is well-established, as testosterone facilitates sexual motivation and performance by acting on central nervous system pathways and peripheral reproductive organs (Genchi et al., 2022). Thus, the significant correlation in this study corroborates the physiological basis of testosterone as a primary driver of libido in bulls.

Conversely, the lack of significant correlation between semen pH and other reproductive parameters aligns with the fact that seminal pH is tightly regulated to maintain sperm viability and motility and is less influenced by short-term fluctuations in hormone levels or nutrition (Dai et al., 2024). The moderate, non-significant correlation between testosterone and semen volume further suggests that, while hormones are important, seminal plasma volume also depends on accessory gland function and the local testicular environment, which are regulated by additional factors beyond systemic testosterone levels (Li et al., 2024).

This study has several limitations. Proximate and phytochemical analyses of the leaf powders were not performed; instead, nutrient adequacy was verified based on previously published proximate data (Bamishaiye et al., 2011; Tenrimega et al., 2024; Pazla et al., 2023). In addition, semen evaluation was restricted to volume and pH, without the inclusion of standard semen quality parameters such as motility, concentration, morphology, or viability. As such, this study should be considered a preliminary investigation aimed at identifying potential effects of phytogenic supplementation on basic reproductive indicators.

Any future study should directly analyze the bioactive compounds in these phytopharmaceutical supplements and include a broader range of sperm quality parameters and molecular biomarkers to gain a more comprehensive understanding of their contribution to reproductive outcomes. Overall, the current findings highlight the potential of Moringa, Gliricidia, and Indigofera leaf powders as viable phytopharmaceutical feed supplements for improving reproductive endocrinology and sperm parameters in Bali bulls. This nutritional strategy also supports Indonesia’s national livestock development agenda, including the Asta Cita Program, by promoting sustainable productivity and food security.

On the other hand, limitations such as small sample size and the absence of detailed phytochemical profiles highlight the need for further study. Any future study should explore dose-response relationships, investigate mechanistic pathways through molecular biomarkers, and evaluate long-term fertility outcomes to optimize practical applications. Additionally, while this study focused on phytogenic leaf powder as a feed supplement, future studies could integrate nutritional interventions with broader livestock sustainability challenges, including parasite management and climate-related stressors, to provide a holistic understanding of reproductive performance in Bali bulls.

CONCLUSION

In conclusion, dietary supplementation with Moringa oleifera, Gliricidia sepium, and Indigofera zollingeriana leaf powders over a 90-day period improved libido, semen volume, and hormonal levels of testosterone and adiponectin in Bali bulls, while semen pH remained within the physiological range. These findings highlight the potential of all three leaf powders as natural feed additives to enhance reproductive performance and support sustainable breeding programs in Bali bulls. However, the results should be interpreted with caution given the small sample size and the absence of a control group. Future studies are therefore needed to optimize supplementation protocols, clarify the underlying mechanisms, and assess long-term reproductive outcomes to strengthen the practical application of these supplements.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the support from the RIIM Competitive Program funded jointly by the National Research and Innovation Agency (BRIN) and LPDP, under Contract Number 00590/UN4.22/PT.01.03/2025. We also thank all members of the Berkah Farms, and the Research Center for Applied Zoology and Animal Husbandry, National Research and Innovation Agency for their invaluable assistance.

Novelty Statement

This study provides new evidence on the comparative effects of three phytogenic leaf powders—Moringa oleifera, Gliricidia sepium, and Indigofera zollingeriana—on reproductive parameters of Bali bulls. It introduces a novel nutritional approach by integrating these tropical phytogenic sources into a standardized diet to assess libido, semen characteristics, and hormonal responses (testosterone and adiponectin). The results highlight a unique association between phytogenic bioactive compounds and the concurrent enhancement of testosterone and adiponectin, indicating a synergistic influence on reproductive and metabolic regulation. These findings offer new perspectives for developing sustainable phytogenic supplementation strategies to improve fertility and breeding efficiency in Bali bulls.

AUTHOR’S CONTRIBUTION

The study was carried out by AN, AMD, ANC, ED, FI, IUA, RR, AASA and all authors contributed equally. In addition, MY, SS, MM, MFA and AMA made essential contributions to the organization and analysis of the data for this work. All authors above consented to be held responsible for all parts of the work and participated in its preparation, drafting, and revision. They also provided final permission for the version that was published.

Generative AI and AI-assisted technology statement

The authors declare that no Generative AI was used in the creation of this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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