The Potential of Lontar Fruit (Borassus flabellifer) Fiber Extract as a Natural Bioactive Compound to Enhance the Quality of Boar Semen

Wilmientje Marlene Nalley1*, Thomas Mata Hine1, Kirenius Uly1, Petrus Kune1, Agustinus Ridlof Riwu1, Ni Made Paramita Setyani1, Yustiany Yuliana Bette1, Alvrado Bire Lawa1, Adyanto Nessy Banamtuan1, Ronny Markus Jami Riwu1, Annisa Rahmi2, Athhar Manabi Diansyah3, Abdullah Baharun2

1Faculty of Animal Husbandry, Marine and Fisheries, Nusa Cendana University, Kupang, Indonesia; 2Department of Animal Science, Faculty of Agriculture, Universitas Djuanda, Bogor, Indonesia; 3Faculty of Animal Science, Hasanuddin University, Makassar, Indonesia.

Received | September 13, 2025; Accepted | October 20, 2025; Published | January 05, 2026

*Correspondence | Wilmientje Marlene Nalley, Faculty of Animal Husbandry, Marine and Fisheries, Nusa Cendana University, Kupang, Indonesia; Email: [email protected]

Citation | Nalley WM, Hine TM, Uly K, Kune P, Riwu AR, Setyani NMP, Bette YY, Lawa AB, Banamtuan AN, Riwu RMJ, Rahmi A, Diansyah AM, Baharun A (2026). The potential of lontar fruit (Borassus flabellifer) fiber extract as a natural bioactive compound to enhance the quality of boar semen. J. Anim. Health Prod. 14(1): 56-64.

DOI | https://dx.doi.org/10.17582/journal.jahp/2026/14.1.56.64

ISSN (Online) | 2308-2801

Copyright: 2026 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

INTRODUCTION

Sperm preservation represents a pivotal technology in contemporary livestock farming, particularly within the context of artificial insemination programs for swine (Waberski et al., 2019). This technique has been demonstrated to facilitate broader genetic distribution, enhanced reproductive efficiency, and diminished costs associated with the maintenance of breeding stock. However, the efficacy of this technology is contingent upon the effectiveness of the sperm diluent employed during storage. In the domain of boar sperm preservation, the utilization of dilution agents such as Tris and Beltsville Thawing Solution (BTS) has emerged as the prevailing standard of practice. This phenomenon is primarily attributed to the efficacy of these agents in preserving the viability and motility of sperm (Namula et al., 2019; Vinansius et al., 2019). Notwithstanding, the primary challenge in sperm storage is increased oxidative stress, which can result in membrane integrity impairment, reduced motility, and diminished sperm fertility (Dutta et al., 2019). To address this challenge, the addition of antioxidant supplements is necessary to help stabilize the physiological conditions of sperm during storage.

Although Tris and BTS diluents have been widely used in sperm preservation, their effectiveness is still limited due to the accumulation of reactive oxygen species (ROS), which contribute to a decline in sperm quality over time (Chianese and Pierantoni, 2021; Mislei et al., 2020). This oxidative stress can cause membrane lipid damage, protein denaturation (Jakubczyk et al., 2020), and DNA fragmentation, (Hamilton and Assumpção, 2020) which ultimately have a negative impact on sperm fertility. Various synthetic antioxidants have been used to address this issue, but their effectiveness remains highly variable. Therefore, this study proposes the use of a natural material, namely the extract of lontar fruit fiber (Borassus flabellifer), as an alternative antioxidant supplement in sperm diluents to improve pig sperm quality during storage.

Borassus flabellifer, commonly known as the palmyra palm, contains significant amounts of phenolic compounds and flavonoids, which exhibit strong antioxidant properties. These compounds are crucial in neutralizing reactive oxygen species (ROS) and reducing oxidative stress, which can damage sperm cells. These include gallic acid, coumarin, and quercetin, which are present in the male flower ethanolic extract of B. flabellifer (Tunit et al., 2022). The extract contains high levels of flavonoids, such as quercetin and epigallocatechin gallate (EGCG), which contribute to its antioxidant activity (Kurpios et al., 2021; Tunit et al., 2022). Studies have shown that B. flabellifer extracts can protect cells from oxidative stress by maintaining cellular redox balance and mitochondrial function (Tusskorn et al., 2021). This is particularly important for sperm cells, which are highly susceptible to oxidative damage.

Tris and BTS diluents have been demonstrated to be effective in maintaining sperm viability and motility. However, both are less effective in counteracting the accumulation of reactive oxygen species (ROS), which causes oxidative stress, plasma membrane damage, decreased motility, and sperm DNA fragmentation. A substantial body of research has previously investigated a variety of natural compounds.

The distinguishing features and added value of this study are attributable to its innovative approach to utilizing local natural materials to enhance reproductive technology (Hamed et al., 2019; Luo et al., 2020; Ahmed et al., 2024). The utilization of lontar fruit fibre extract as a supplement in Tris and BTS diluents provides a safer and more sustainable natural solution in comparison to synthetic antioxidants. Furthermore, this study makes a substantial contribution to the field of reproductive biotechnology and supports the utilization of abundant yet underutilized biological resources (Mahayasa et al., 2024). Consequently, the findings of this study are anticipated to offer substantial scientific contributions and implications for enhancing the efficiency of the livestock industry through the optimization of sperm preservation technology.

The objective of this study is to compare the effectiveness of Lontar fruit fiber extract with conventional diluents in maintaining sperm viability, motility, and membrane integrity. Furthermore, this study will ascertain the optimal concentration of Lontar fruit fiber extract that provides the most efficacious protective effect on sperm during storage.

MATERIALS AND METHODS

Experimental design and ethical approval

All procedures were approved by the Animal Ethics Commission at the Faculty of Animal Husbandry, Marine and Fisheries, Nusa Cendana University (075/1.KT/KEPPKP/IV/2024). Animal handling, semen collection, and laboratory analyses complied with institutional and national guidelines for the ethical use of animals in research. The experiment was conducted using a completely randomized factorial design (2 × 5). Factor A was the semen diluent (Tris–fructose–citric acid [Tris] vs. Beltsville Thawing Solution [BTS]), and Factor B was the concentration of Lontar fruit fiber extract (LFFE; 0, 3, 6, 9, and 12% w/v). Each treatment combination was replicated five times, resulting in a total of 50 ejaculates (experimental units) collected from three clinically healthy 3-year-old Landrace boars maintained under standard husbandry conditions. Only ejaculates meeting the inclusion criteria (>80% progressive motility and <20% morphological abnormalities, in line with the Indonesian National Standard for artificial insemination) were used in the experiment.

Preparation of lontar fruit fiber extract (LFFE)

Ripe Borassus flabellifer fruit fibers (dark yellow) were trimmed, homogenized, and macerated (using ethanol 70%) in distilled water (1:10, w/v) for 72 h at room temperature with intermittent agitation. The suspension was filtered through cloth or filter paper, and the filtrate was dried at 40 °C in an incubator to obtain LFFE powder. Working solutions at the desired concentrations were freshly prepared on the day of use.

Semen dilution and preservation

Qualified ejaculates were diluted with the assigned extender at a 1:3 semen-to-diluent ratio according to treatment allocation. Diluted samples were stored at 15–20 °C, following short-term boar semen preservation protocols. Sperm quality assessments were carried out at 0 h and at 12-h intervals up to 96 h.

Semen quality evaluation

Sperm motility was assessed under a light microscope at 400× magnification from eight randomly selected microscopic fields. Viability was evaluated using the eosin–nigrosin staining technique, in which at least 200 sperm cells per sample were observed; viable sperm excluded the stain, while non-viable cells appeared pink (Alfian et al., 2025). Morphological assessment was carried out on stained smears by examining a minimum of 200 sperm cells per sample, with abnormalities recorded in the head, midpiece, tail, or in the presence of cytoplasmic droplets (Nirmala et al., 2025). Plasma membrane integrity was evaluated using the Hypo-Osmotic Swelling Test (HOST), where intact membranes were indicated by characteristic tail swelling in a hypotonic solution (Diansyah et al., 2025). In addition, sperm concentration was measured at baseline using a hemocytometer or an equivalent validated method to confirm sample quality prior to dilution.

Statistical analysis

Data were analyzed using linear mixed-effects models, with diluent, LFFE concentration, storage time, and their interactions as fixed effects. Ejaculate (nested within boar) was included as a random effect to account for repeated measures. Model assumptions were verified, and data were transformed when necessary. Tukey’s test was used for post-hoc multiple comparisons. Results are expressed as mean ± standard deviation (SD), with significance set at p < 0.05. Statistical analyses were performed in SPSS v25.0 (IBM Corp., Armonk, NY, USA).

RESULTS

Sperm motility

As shown in Table 1, at 0 hours of storage, no significant differences (P > 0.05) in sperm motility were observed among treatments. However, from 12 hours onward, significant differences emerged (P < 0.05). The highest motility was consistently recorded in treatments supplemented with 9% LFFE (P4 and P9), with values of 48.00% and 47.60% at 60 hours, respectively. These were significantly higher than the control (P1, 20.00%) and other treatments. Lower or higher LFFE concentrations showed reduced motility, indicating 9% as the most effective level for maintaining sperm motility during storage.

Sperm viability

The incorporation of LFFE into Tris t and BTS diluent at varying concentrations (0%, 3%, 6%, 9%, and 12%) did not demonstrate a substantial impact (P > 0.05) on sperm motility at hour 0 of storage (following dilution). However, beginning at the 12th hour of storage, a substantial increase (P<0.05) in sperm motility was observed in the treatment with 9% LFFE addition, both in Tris and BTS diluent, in comparison to the other treatments (Table 2).

 

Table 1: Percentage of boar sperm motility during storage up to 60 hours in Tris and BTS extenders supplemented with different levels of LFFE.

Treatment	Storage times (hours)
	0	12	24	36	48	60
P1	84.00±2.00a	70.00±7.07bc	60.00±5.00c	49.00±6.52c	33.00±4.47e	20.00±3.54g
P2	83.00±2.00a	73.00±2.74abc	61.00±2.24c	52.00±2.74c	38.00±2.74d	26.00±4.18ef
P3	83.00±2.00a	73.00±2.74abc	63.00±2.74bc	51.00±2.24c	40.00±3.54cd	29.00±4.18de
P4	83.00±2.00a	78.00±2.74a	73.00±2.74a	68.00±2.74a	61.00±5.48a	48.00±2.74a
P5	83.00±2.00a	75.00±0.00ab	68.00±2.74ab	60.00±0.00b	50.00±0.00b	40.00±0.00b
P6	83.00±2.00a	69.00±5.48c	55.00±3.54d	42.00±2.74d	33.00±2.74e	23.00±2.74fg
P7	83.00±2.00a	71.00±2.24bc	62.00±2.74c	52.00±2.74c	40.80±1.10cd	30.00±0.00cde
P8	83.00±2.00a	70.00±3.54bc	62.00±4.47c	52.00±4.47c	43.00±2.74c	33.00±2.74cd
P9	83.00±2.00a	78.00±2.74a	73.00±2.74a	63.00±2.74b	58.00±2.74a	47.60±2.51a
P10	83.00±2.00a	75.00±5.00ab	65.00±7.07bc	54.00±5.48c	44.00±5.48c	34.00±5.48c
P-value	1.000	0.003	0.00	0.00	0.00	0.00

 

Note: Different superscript letters within the same column indicate significant differences among treatments (p < 0.05, Tukey’s test). Tris treatments are represented by P1–P5, and BTS treatments by P6–P10, each corresponding to different levels of LFFE supplementation.

 

Table 2: Percentage of boar sperm viability during storage up to 60 hours in Tris and BTS extenders supplemented with different levels of LFFE.

Treatment	Storage time (hours)
	0	12	24	36	48	60
P1	89.54±2.37a	79.63±3.43bcde	66.37±4.87c	56.68±5.97de	39.11±4.44e	30.22±4.77f
P2	89.44±2.55a	77.99±2.64cde	67.03±4.08c	58.30±2.56d	46.62±2.89cd	35.42±4.00def
P3	89.00±2.03a	82.08±1.18abcd	69.73±3.54bc	59.09±1.90cd	48.24±4.80cd	39.01±5.44cde
P4	90.40±1.67a	86.46±1.76a	80.30±1.28a	77.90±1.20a	68.73±6.85a	60.06±3.50a
P5	89.32±2.10a	83.44±0.78abc	77.21±3.04a	66.66±7.24bc	59.64±2.66b	47.05±3.36b
P6	89.60±2.50a	75.93±7.19e	63.67±3.94c	50.31±4.98e	42.74±4.73de	34.58±4.45ef
P7	89.37±2.63a	78.53±3.99cde	70.05±4.66bc	59.19±3.58cd	48.19±2.56cd	38.19±3.98cde
P8	88.85±2.20a	76.82±5.12de	68.90±8.94bc	57.83±7.53d	50.60±3.33c	42.66±6.00bc
P9	90.03±1.92a	85.17±3.29ab	80.65±4.10a	71.08±4.43ab	66.52±4.81a	56.15±4.03a
P10	89.46±1.85a	82.31±5.22abcd	74.71±7.18ab	64.18±9.56bcd	52.41±9.97c	41.85±8.74bcd
P-value	0.991	0.001	0.00	0.00	0.00	0.00

 

Note: Different superscript letters within the same column indicate significant differences among treatments (p < 0.05, Tukey’s test). Tris treatments are represented by P1–P5, and BTS treatments by P6–P10, each corresponding to different levels of LFFE supplementation.

 

Table 3: Percentage of boar sperm abnormality during storage up to 60 hours in Tris and BTS extenders supplemented with different levels of LFFE.

Treatment	Storage time (hours)
	0	12	24	36	48	60
P1	4.02±0.43a	4.26±0.37ab	4.31±0.29a	4.54±0.35a	4.81±0.31a	5.09±0.28a
P2	3.97±0.48a	4.21±0.56ab	4.49±0.59a	4.93±0.65a	5.06±0.60a	5.29±0.61a
P3	3.92±0.59a	4.08±0.60ab	4.36±0.59a	4.57±0.57a	4.82±0.50a	5.01±0.53a
P4	3.91±0.60a	4.02±0.55a	4.24±0.60a	4.71±0.72a	5.01±0.63a	5.31±0.69a
P5	3.93±0.54a	4.15±0.63ab	4.43±0.70a	4.82±0.77a	5.12±0.72a	6.46±0.48b
P6	3.93±0.57a	5.11±0.58b	6.11±0.80b	7.72±0.49cd	9.25±0.54d	10.96±0.80d
P7	4.01±0.64a	4.88±0.86ab	6.39±1.24b	8.18±1.19d	8.76±0.72cd	10.89±0.49d
P8	4.05±0.62a	4.94±0.88ab	5.78±0.80b	7.03±0.94bc	8.02±1.01bc	9.73±0.98c
P9	4.15±0.56a	4.72±0.95ab	5.52±0.83b	6.51±1.36b	7.40±0.59b	7.50±1.51b
P10	4.25±0.72a	5.09±0.83b	5.92±0.45b	6.50±0.41b	7.22±0.38b	9.27±1.05c
P-value	0.995	0.070	0.00	0.00	0.00	0.00

 

Note: Different superscript letters within the same row indicate significant differences among treatments (p < 0.05, Tukey’s test). Tris treatments are represented by P1–P5, and BTS treatments by P6–P10, each corresponding to different levels of LFFE supplementation.

 

This increase in sperm motility persisted until the 24th, 36th, 48th, and 60th hours of storage. At the 60th hour, the highest sperm motility was recorded in the group with 9% LFFE addition, at 48.00% for Tris diluent and 47.60% for BTS diluent. This was significantly different (P<0.05) compared to the control group and other treatment groups with different LFFE concentrations.

Sperm abnormality

A similar pattern was also observed in sperm viability. Abnormality in Table 3 showed no statistically significant differences (P > 0.05) were observed between the treatments at 0 hours of storage. However, beginning at 12 hours of storage, the incorporation of 9% LFFE demonstrated a substantial enhancement in sperm viability (P<0.05) in comparison to alternative treatments, across both Tris and BTS dilutions.

At the 60th hour of storage, the highest sperm viability was achieved by the treatment group with 9% LFFE addition, at 60.06% in Tris diluent and 56.15% in BTS diluent. The values obtained in this study differed significantly (P<0.05) from the control group and the treatment groups with other LFFE concentrations.

Plasma membrane integrity

The evaluation of sperm plasma membrane integrity (PMI) yielded results that were consistent with those of other parameters. No statistically significant differences (P > 0.05) were observed at 0 hours of storage (Table 4).

 

Table 4: Percentage of boar sperm plasma membrane integrity during storage up to 60 hours in Tris and BTS extenders supplemented with different levels of LFFE.

Treatment	Storage time (Hours)
	0	12	24	36	48	60
P1	87.85±2.38a	79.16±3.57cd	66.83±5.09de	57.39±5.47de	32.78±16.43e	32.54±2.02e
P2	88.05±2.86a	78.34±2.49cd	69.38±6.08cde	58.63±2.58d	47.05±3.44d	35.84±4.20de
P3	88.46±2.96a	80.94±2.28bcd	70.02±3.57cde	61.71±4.47d	50.72±5.02d	39.99±5.06cd
P4	88.43±2.53a	85.88±1.46a	80.22±1.06a	78.21±1.75a	70.46±5.40a	60.39±3.09a
P5	88.50±2.72a	81.36±0.87abc	77.54±2.44ab	69.38±6.25bc	61.14±1.53bc	47.48±2.74b
P6	86.96±2.03a	76.28±5.42d	64.95±2.69e	50.83±3.87e	45.19±3.26d	36.36±4.88de
P7	87.25±2.46a	79.77±3.42bcd	71.43±4.26bcde	60.03±3.54d	49.33±1.77d	40.86±3.43cd
P8	88.33±2.57a	78.92±3.78cd	72.81±8.14bcde	59.85±6.14d	52.34±2.79cd	45.03±5.10bc
P9	89.06±3.11a	84.31±2.13ab	80.93±2.98a	71.58±3.83ab	67.28±4.34ab	56.07±3.71a
P10	87.85±2.38a	79.16±3.57cd	66.83±5.09de	57.39±5.47de	32.78±16.43e	32.54±2.02e
P-value	0.977	0.002	0.00	0.00	0.00	0.00

 

Note: Different superscript letters within the same column indicate significant differences among treatments (p < 0.05, Tukey’s test). Tris treatments are represented by P1–P5, and BTS treatments by P6–P10, each corresponding to different levels of LFFE supplementation.

 

However, beginning at 12 hours of storage, a substantial increase (P<0.05) in MPU was observed in the group with 9% LFFE addition, both in Tris and BTS diluents.

At the 60th hour of storage, the highest percentage of sperm with PMI was recorded in the 9% LFFE group, at 60.39% for Tris diluent and 56.07% for BTS diluent. The values obtained in this study differed significantly (P < 0.05) from the control group and the treatment groups with other LFFE concentrations.

DISCUSSION

Lontar fruit fibre extract is a natural source that is abundant in bioactive compounds. These bioactive compounds have the potential to improve sperm quality during storage. As indicated by the findings of preceding studies, the lontar fruit (Borassus flabellifer) contains a variety of significant compounds, including phenolic compounds, flavonoids, vitamin C (Lenggu et al., 2020), and dietary fibre with comparatively high antioxidant activity. These compounds neutralize ROS, reduce oxidative damage, and maintain cellular health, making B. flabellifer a promising natural agent for enhancing sperm quality and reproductive health (Dzigbor et al., 2025). Excessive ROS concentrations have been demonstrated to trigger a series of deleterious effects, including plasma membrane damage, lipid peroxidation, and mitochondrial dysfunction (Darmawan, 2007). These effects can culminate in reduced sperm motility and viability. Consequently, the inherent antioxidant properties of LFFE function as a biological shield, thereby preserving the structural and functional integrity of sperm cells. The present study demonstrates that the incorporation of 9% LFFE, both in Tris and BTS diluent, results in enhanced motility, viability, IPM, and a reduction in sperm abnormalities.

In addition to its antioxidant content, Lontar fruit is a source of complex carbohydrates, including polysaccharides and water-soluble fiber. These carbohydrates have the potential to serve as an additional energy source for sperm during storage (Aimanah, 2018). Sperm are known to require substantial energy to maintain optimal motility, membrane integrity, and fertilization capacity. This energy requirement is well-documented and well-established. This phenomenon is particularly evident under conditions of long-term storage, which can result in a decline in intracellular energy reserves. The complex carbohydrates present in LFFE, notably in the form of simple sugars resulting from the hydrolysis of dietary fiber, can function as an alternative metabolic substrate. This, in turn, supports mitochondrial activity and extends the progressive motility of sperm during storage (Hine et al., 2014).

The combined support of antioxidant compounds and carbohydrates in LFFE is a key factor explaining why the addition of LFFE at an optimal level of 9% significantly improves the quality of boar sperm after 60 hours of storage. Consequently, LFFE functions not only as a protective compound against oxidative stress but also as an essential natural energy source for the maintenance of sperm physiological function. These findings underscore the efficacy and applicability of LFFE as a natural additive in semen diluent formulations, thereby opening avenues for the innovative utilization of locally sourced products to enhance the efficiency of livestock reproductive technology.

The findings of this study suggest that both Tris and BTS diluent possess the capacity to preserve the integrity of boar sperm during storage, particularly when employed in conjunction with the incorporation of LFFE at a concentration of 9%. However, studies have demonstrated that the utilization of Tris diluent generally results in sperm of superior quality when compared to BTS. This is evidenced by parameters such as motility, viability, the presence of abnormalities, and the integrity of the sperm plasma membrane, particularly following 60 hours of storage. The findings of this study are consistent with the fundamental characteristics of each diluent, which have been extensively documented in the extant literature. Tris is a tris (hydroxymethyl) aminomethane-based diluent with excellent buffering capacity. Research findings have demonstrated that this compound can maintain pH stability within the semen environment during storage (Blegur et al., 2020; Hine and Nalley, 2025). As is well-established, pH stability is imperative for maintaining plasma membrane integrity, as well as for ensuring the optimal level of enzymatic activity and ion balance in sperm. Consequently, this results in a physiological slowing down of sperm quality degradation during storage.

In contrast, BTS has gained significant popularity in the swine artificial insemination industry as a practical diluent, primarily due to its ease of use and its ability to maintain sperm quality during short to medium-term storage (Nalley et al., 2024). However, the buffering capacity and membrane protection of BTS are comparatively limited. The discrepancy in effectiveness between the two types of extenders becomes increasingly evident under storage conditions that induce oxidative stress, where the contribution of LFFE bioactive compounds functions as an ancillary protective factor. Consequently, while the use of LFFE has been demonstrated to enhance sperm quality in both types of diluents, the optimization of Tris diluent usage is still advised for boar semen storage applications, particularly in scenarios involving storage periods exceeding 48 hours. The combination of Tris diluent with LFFE at optimal levels could serve as a potential strategy to enhance the efficiency of boar artificial insemination programs through improved sperm quality during storage.

The findings of this study carry substantial practical ramifications for the advancement of swine reproductive technology, particularly regarding enhancing the efficiency and success of artificial insemination programs. A significant challenge in implementing artificial insemination in swine agriculture pertains to the temporal limitations imposed on the storage of fresh semen, which can result in a decline in sperm quality (Hine and Nalley, 2025). This decline encompasses parameters such as motility, viability, PMI, and an increase in abnormalities, thereby exerting a direct influence on the success rates of fertilization. The results of this study indicate that the addition of 9% Lontar fruit fiber extract to Tris or BTS diluents can significantly maintain boar sperm quality for up to 60 hours of storage. This finding indicates the possibility of LFFE functioning as an effective natural additive. It demonstrates the capacity to prolong the storage life of fresh semen while decreasing the necessity for synthetic additives commonly employed in the boar farming industry.

In addition, the utilization of local materials, such as LFFE, possesses the capacity to furnish a more economical and accessible solution for farmers, particularly in tropical regions such as Indonesia. In this country, the Lontar palm (Borassus flabellifer) is widely prevalent but has not yet been fully utilized to its full potential. The employment of LFFE in semen diluents offers a multifaceted strategy for addressing contemporary agricultural challenges. Firstly, the product under consideration provides an alternative nutritional supplement that has the potential to enhance sperm performance. Secondly, the practice aligns with the principles of sustainability that have emerged in modern farming systems. It achieves this alignment by leveraging the local biological potential to improve production efficiency. Consequently, the findings of this study provide a significant scientific contribution to the understanding of the mechanism of action of natural bioactive compounds on sperm quality. Moreover, the results of this study have a substantial impact on the promotion of innovative, cost-effective, and environmentally friendly reproductive technologies in the swine farming industry.

Even though the present study provides relevant information regarding the prospective use of LFFE as a natural additive to enhance the quality of boar sperm in both Tris and BTS diluent, it is imperative to acknowledge the presence of several methodological and technical limitations. A notable limitation of the present study is the absence of phytochemical analysis, which is a critical component of evaluating bioactive compounds in LFFE associated with improved sperm quality.

However, a more comprehensive understanding of the chemical composition of LFFE, including the types and concentrations of flavonoids, phenolics, polysaccharides, and other antioxidants, is crucial to elucidating the biological mechanisms underlying the protective effects of LFFE on sperm cells. Absent such data, the interpretation of the results remains general and cannot be fully attributed to specific active compounds contained in LFFE. Furthermore, the study’s design was constrained to the assessment of sperm quality parameters in vitro, including motility, viability, abnormalities, and PMI. It did not encompass the implementation of fertility assessments in the field, such as the measurement of pregnancy success rates or birth rates after artificial insemination utilizing semen that had undergone treatment with LFFE. However, while in vitro sperm quality parameters are indeed significant, they do not always directly correlate with actual fertility outcomes in the field. Consequently, to ensure the practical effectiveness of LFFE use in artificial insemination programs, further testing at the farm level involving real fertility parameters is required.

A notable constraint of the present study is its scope, which is currently confined to a single species, specifically boars, and utilizes semen from a particular breed. The potential of LFFE as a natural semen diluent additive in other livestock species, such as cattle, goats, or sheep, as well as under various environmental conditions, has not yet been explored. Furthermore, the interaction between LFFE and other diluents beyond Tris and BTS remains to be investigated. This presents a potential avenue for further research, which could lead to the development of more applicable, comprehensive, and specific recommendations tailored to the diverse range of farm conditions.

In consideration of the findings and the constraints that have been identified, the present study suggests several avenues for future research. These avenues include the potential of LFFE as a natural semen diluent additive. A primary recommendation is the necessity of a comprehensive phytochemical characterization of LFFE, with the aim of identifying the types, levels, and stability of bioactive compounds, including phenolics, flavonoids, polysaccharides, vitamin C, and other antioxidant compounds (Prasad et al., 2022, 2023; Bahera and Nayak, 2022). These bioactive compounds are suspected to contribute to the enhancement of sperm quality. This analysis can be conducted using high-performance liquid chromatography (HPLC) or spectrophotometry. It is imperative to ensure that the resulting data is not only descriptive but also capable of explaining the molecular mechanisms underlying the positive effects of LFFE.

Moreover, further experimentation is necessary to assess the repercussions of implementing LFFE in semen diluents on real fertility parameters in the field. The following parameters are of relevance in this context: artificial insemination success rates, pregnancy rates, live births, and the quality of offspring produced. Such evaluations are imperative to ascertain that the observed enhancements in sperm quality in vitro genuinely exert a favourable influence on reproductive efficiency at the livestock population level. This recommendation assumes particular significance considering the study’s findings, which indicate that Tris diluent augmented with 9% LFFE can preserve sperm quality for a duration of up to 60 hours of storage. To ensure the validity of this assertion, it is imperative that the findings be substantiated through field testing.

Furthermore, additional exploration of LFFE’s potential in other livestock species, including cattle, goats, and sheep, is imperative to assess the consistency of this compound’s efficacy across diverse sperm types, which possess varying physiological characteristics. These studies are of particular importance because each species exhibits different sensitivities to storage stress and to additive components in diluents. In addition, research initiatives that concentrate on the incorporation of LFFE with other natural additives, including honey, herbal extracts, and micronutrient supplements, are justified. The objective of these research endeavours is to formulate a semen diluent that is more optimal, efficient, and environmentally friendly.

It is imperative that further research be conducted to assess the feasibility of implementing LFFE in more stable and pragmatic processed forms, such as dry extracts or microcapsules, to facilitate its on-farm application. In addition, future work should include on-farm AI trials, multi-breed semen testing, and antioxidant mechanism assays. Collectively, these efforts would advance LFFE-based innovations that not only provide scientific benefits but also enhance reproductive technology efficiency and support sustainable livestock production systems utilizing local resources.

CONCLUSION

Supplementation of 9% LFFE effectively preserves sperm motility, viability, and PMI up to 60 h. LFFE is a potential natural additive for semen storage. These results suggest that LFFE has the potential to act as a natural additive, thereby enhancing the storage life and quality of boar semen. It is recommended that Lontar fruit fiber extract be used as a natural additive in boar semen diluents, particularly at a level of 9% in Tris diluent. Further research is necessary to test its effectiveness on field pregnancy success rates and its potential application in other livestock species.

ACKNOWLEDGEMENTS

The authors would like to express their sincere gratitude to the Head and laboratory staff of the Faculty of Animal Husbandry, Marine and Fisheries, Nusa Cendana University, for providing the necessary facilities and technical support throughout the course of the study.

Novelty Statement

This study is the first to systematically evaluate the use of Lontar fruit (Borassus flabellifer) fiber extract (LFFE) as a natural bioactive additive in both Tris and Beltsville Thawing Solution (BTS) extenders for boar semen preservation. The findings demonstrate that supplementation with 9% LFFE significantly improves sperm motility, viability, and plasma membrane integrity while decreasing abnormalities during storage for up to 60 hours. This represents a novel, eco-friendly approach, utilizing an abundant, underexploited local biological resource to enhance swine reproductive technology and artificial insemination success.

AUTHOR’S CONTRIBUTION

WMN, TMH, KU and PK: Conceived and designed the experiment. WMN, TMH, KU and PK: Performed the experimental procedures. WMN, ARR, NMPS, and YYB: Supervised and coordinated the research and provided clinical data. ABL and ANB: Statistical analysis was conducted. RMJR, AR, AMD, and AB: The initial draft of the manuscript was prepared. All authors critically reviewed and approved the final version of the manuscript.

Generative AI and AI-assisted technology statement

No generative AI or AI-assisted technology was utilized in the design, data collection, analysis, or initial preparation of this manuscript. All experimental design, data processing, and preliminary drafting were conducted exclusively by the authors without the involvement of generative AI. However, DeepL and Quillbot, both language-support software tools, were used to improve the linguistic quality of the manuscript. These tools were used for editing, grammar correction, and paraphrasing to enhance readability, while ensuring that all original ideas scientific interpretations remain the authors’ own.

Conflict of interest

The authors have declared no conflict of interests.

REFERENCES

Ahmed H, Ijaz MU, Riaz M, Ullah F, Samir H, Shuaib M, Swelum AA (2024). Taxifolin: A flavonoid in the freezing medium augments post-thaw semen quality and in vivo fertility potential of buffalo bull spermatozoa. Cryobiology, 117: 104980. https://doi.org/10.1016/j.cryobiol.2024.104980

Aimanah U (2018). Karakteristik pembuatan manisan buah lontar dalam penyimpanan suhu dingin: Characteristics of sweet lontar fruit making in cold temperature storage. J Agrisistem, 14(2): 157–163. Available at: https://www.ejournal.polbangtan-gowa.ac.id/index.php/J-Agr/article/view/39

Alfian AM, Yusuf M, Ako A, Toleng AL, Diansyah AM, Amrullah MF, Rahmat R, Hasrin H (2025). The effects of mineral mix supplementation on reproductive performance of Bali bulls. Egypt J. Vet. Sci., pp. 1–8. https://doi.org/10.21608/ejvs.2025.335655.2502

Behera S, Nayak B (2022). Phytochemical constituents and nutritional potential of palmyra palm: A review. Rev. Contemp. Sci. Acad. Stud., 2. https://doi.org/10.55454/rcsas.2.12.2022.003

Blegur J, Nalley W, Hine TM (2020). Pengaruh penambahan virgin coconut oil dalam pengencer tris kuning telur terhadap kualitas spermatozoa sapi bali selama preservasi. J. Nukleus Peternakan, 7(2): 130–138. https://doi.org/10.35508/nukleus.v7i2.2997

Chianese R, Pierantoni R (2021). Mitochondrial reactive oxygen species (ROS) production alters sperm quality. Antioxidants, 10(1): 92. https://doi.org/10.3390/antiox10010092

Darmawan H (2007). Production of ROS and its effects on mitochondrial and nuclear DNA, human spermatozoa, and sperm function. Med. J. Indones., 16(2): 127–133. https://doi.org/10.13181/mji.v16i2.268

Diansyah AM, Yusuf M, Rahmat R, Alfian AM (2025). Enhanced sperm sexing efficiency and quality preservation in Bali bulls using freeze-dried albumin separation media. Open Vet. J., 15(6): 2416–2426. https://doi.org/10.5455/OVJ.2025.v15.i6.14

Dutta S, Majzoub A, Agarwal A (2019). Oxidative stress and sperm function: A systematic review on evaluation and management. Arab J. Urol., 17(2): 87–97. https://doi.org/10.1080/2090598X.2019.1599624

Dzigbor A, Neglo D, Dzah CS, Sraha R (2025). Total phenolic content, phytochemical screening, antioxidant and antimicrobial activities of Borassus flabellifer and Borassus aethiopum fruits. Food Chem. Adv., 7: 100937. https://doi.org/10.1016/j.focha.2025.100937

Hamed YS, Abdin M, Akhtar HMS, Chen D, Wan P, Chen G, Zeng X (2019). Extraction, purification by macrospores resin and in vitro antioxidant activity of flavonoids from Moringa oliefera leaves. S. Afr. J. Bot., 124: 270–279. https://doi.org/10.1016/j.sajb.2019.05.006

Hamilton TRDS, Assumpção MEO (2020). Sperm DNA fragmentation: causes and identification. Zygote, 28(1): 1–8. https://doi.org/10.1017/S0967199419000595

Hine TM, Burhanuddin, Marawali A (2014). Efektivitas air buah lontar dalam mempertahankan motilitas, viabilitas dan daya tahan hidup spermatozoa sapi bali. J. Vet., 15(2): 263–273. Retrieved from https://www.cabidigitallibrary.org/doi/pdf/10.5555/20143294873

Hine TM, Nalley WM (2025). Improving Landrace pig sperm motility and kinematics by adding citrate and glucose to coconut water-egg yolk diluent: A CASA study. J. Adv. Vet. Res., 15(3): 289-293. Retrieved from https://advetresearch.com/index.php/AVR/article/view/2182

Jakubczyk K, Dec K, Kałduńska J, Kawczuga D, Kochman J, Janda K (2020). Reactive oxygen species – sources, functions, oxidative damage. Pol. Merkuriusz Lek, 48(284): 124–127. Retrieved from https://pubmed.ncbi.nlm.nih.gov/32352946/

Kurpios-Piec D, Majewska-Wierzbicka M, Czeczot H, Podsiad M (2021). Flavonoids as hydroxyl radical scavengers, iron reductants and chelators: In vitro antioxidant action. Acta Poloniae Pharm-Drug Res., 78(5): 635-648. https://doi.org/10.32383/appdr/143094

Lenggu CKL, Indriarini D, Amat ALS (2020). Uji aktivitas ekstrak etanol kulit daging buah lontar (Borassus flabellifer Linn) terhadap pertumbuhan Escherichia coli secara in vitro. Cendana Med. J., 8(2): 96–107.

Luo Q, Zhang JR, Li HB, Wu DT, Geng F, Corke H, Wei XL, Gan RY (2020). Green extraction of antioxidant polyphenols from green tea (Camellia sinensis). Antioxidants, 9(9): 785. https://doi.org/10.3390/antiox9090785

Mahayasa NW, Mahayasih PG, Kaldicson A, Achmad AS, Rahayu ST (2024). Effect of extraction methods on the antioxidants and alpha-glucosidase inhibitory activity of Borassus flabellifer L. fruit fiber. Trop. J. Nat. Prod. Res., 8(5). https://doi.org/10.26538/tjnpr/v8i5.2

Mislei B, Bucci D, Malama E, Bollwein H, Mari G (2020). Seasonal changes in ROS concentrations and sperm quality in unfrozen and frozen-thawed stallion semen. Theriogenology, 144: 89–97. https://doi.org/10.1016/j.theriogenology.2019.12.016

Nalley WM, Marawali A, Uly K, Kune P, Telupere F, Setyani NMP, Hine TM (2024). Perbandingan kualitas sperma babi Landrace dalam pengencer Beltsville Thawing Solution, Mulberry III, dan Sperm Life yang ditambahkan ekstrak etanol daun kelor. J. Vet., 25(3): 345-356. https://doi.org/10.19087/jveteriner.2024.25.3.345

Namula Z, Tanihara F, Wittayarat M, Hirata M, Nguyen NT, Hirano T, Le QA, Nii M, Otoi T (2019). Effects of Tris (hydroxymethyl) aminomethane on the quality of frozen-thawed boar spermatozoa. Acta Vet. Hung., 67(1): 106–114. https://doi.org/10.1556/004.2019.012

Nirmala A, Toleng AL, Yusuf M, Herdis H, Diansyah AM, Amrullah MF, Rahmat R, Rajamuddin R, Alfian AM, Hasrin H (2025). Effect of adding kasumba turate (Carthamus tinctorius L.) extract in tris-egg yolk diluent on sperm quality in Bali bulls. J. Anim. Health Prod., 13(1): 154–159. https://doi.org/10.17582/journal.jahp/2025/13.1.154.159

Prasad AB, Arunkumar A, Vignesh S, Chidanand DV, Baskaran N (2022). Exploring the nutritional profiling and health benefits of Palmyra palm haustorium. S. Afr. J. Bot., 151: 228–237. https://doi.org/10.1016/j.sajb.2022.01.027

Prasad AB, Vignesh S, Elumalai A, Anandharaj A, Chidanand DV, Baskaran N (2023). Nutritional and pharmacological properties of palmyra palm. Food Hum., 1: 817–825. https://doi.org/10.1016/j.foohum.2023.07.030

Tunit P, Thammarat P, Okonogi S, Chittasupho C (2022). Hydrogel Containing Borassus flabellifer L. male flower extract for antioxidant, antimicrobial, and anti-inflammatory activity. Gels, 8(2): 126. https://doi.org/10.3390/gels8020126

Tusskorn O, Pansuksan K, MacHana K (2021). Borassus flabellifer L. crude male flower extracts alleviate cisplatin-induced oxidative stress in rat kidney cells. Asian. Pasific J. Trop. Biomed., 11(2): 81-88. https://doi.org/10.4103/2221-1691.303607

Vinansius FW, Nurul I, Trinil S (2019). The quality of Landrace pig spermatozoa during the cold storage process using BTS and CEP-3 diluents added 10% egg yolk at temperatures of 2–5 °C. Russ. J. Agric. Socio-Econ. Sci., 88(4): 223–228. https://doi.org/10.18551/rjoas.2019-04.28

Waberski D, Riesenbeck A, Schulze M, Weitze KF, Johnson L (2019). Application of preserved boar semen for artificial insemination: Past, present and future challenges. Theriogenology, 137: 2–7. https://doi.org/10.1016/j.theriogenology.2019.05.030