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Veterinary research communications2026; 50(5); 482; doi: 10.1007/s11259-026-11424-9

The effect of fenugreek (Trigonella foenum-graecum) on stallion spermatozoa motility and vitality in vitro.

Abstract: This study evaluated the effects of Trigonella foenum-graecum L. (fenugreek) seed extract on stallion spermatozoa quality in vitro, focusing on motility, vitality, and functional competence. In vitro spermatozoa analyses showed that fenugreek seed extract did not demonstrate overt cytotoxicity under the tested conditions: S1 = 1562 µg/mL; S2 = 781 µg/mL; S3 = 390.5 µg/mL; S4 = 195.25 µg/mL; S5 = 97.62 µg/mL; S6 = 48.81 µg/mL; S7 = 24.40 µg/mL. Computer-assisted semen analysis (CASA) revealed a concentration- and time-dependent Treatment × Time interaction for several kinetic parameters. Total motility declined similarly across all groups over incubation time and was not significantly affected by extract concentration, whereas progressive motility, curvilinear velocity, beat-cross frequency, and amplitude of lateral head displacement were selectively increased at specific concentrations (mainly S4 and S7) at the last time point. Metabolic activity (MTT assay) increased significantly relative to the positive control across most concentrations, especially at early incubation time points, with fewer concentrations remaining elevated later. Sperm non-viability (eosin-nigrosin staining) and proAKAP4 levels were also significantly influenced by treatment in a non-monotonic manner: the highest concentration (S1) showed lower non-viability; similarly, the highest concentrations (S1-S3) showed reduced proAKAP4, whereas intermediate-to-lower concentrations showed higher non-viability (mainly S3, S4, S6, and S7) and increased proAKAP4 (mainly S4, S6, and S7), relative to the positive control. Physicochemical parameters of the extract (pH, osmolality) remained within physiological ranges, excluding osmotic or pH-related effects. Fenugreek seeds showed measurable antioxidant activity, with the ABTS assay showing the highest radical-scavenging capacity (10.67 ± 0.28 µmol Trolox equivalents/g dry weight). These findings indicate that fenugreek extract exerts selective, concentration- and time-dependent modulatory effects on stallion sperm function in vitro.
© 2026. The Author(s).
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Publication Date: 2026-07-24 PubMed ID: 42496799PubMed Central: PMC13400685DOI: 10.1007/s11259-026-11424-9Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This study investigates how fenugreek (Trigonella foenum-graecum) seed extract affects the motility, vitality, and overall functional quality of stallion sperm cells in a controlled laboratory setting.
  • The research demonstrates that fenugreek seed extract can selectively enhance certain aspects of sperm motility and metabolic activity without causing toxicity, depending on the concentration and exposure time.

Introduction and Objectives

  • The research aimed to evaluate the in vitro effects of fenugreek seed extract on stallion spermatozoa quality parameters, including motility, vitality, metabolic activity, and functional competence.
  • Fenugreek is known for various bioactive compounds, and here the focus was to see if it can improve sperm function, which could have applications in animal breeding and fertility treatments.

Experimental Conditions and Concentrations

  • Seven concentrations of fenugreek seed extract were tested, labeled S1 through S7, with concentrations ranging from 1562 µg/mL (S1) down to 24.40 µg/mL (S7).
  • The team verified that the physicochemical properties such as pH and osmolality of these extracts remained within physiological norms, ensuring any effects are not due to environmental stress like osmotic imbalance or pH changes.
  • These concentrations were chosen to examine both potential toxic effects and beneficial modulatory effects.

Assessment Techniques

  • Computer-Assisted Semen Analysis (CASA): to objectively measure sperm motility parameters like total motility, progressive motility, curvilinear velocity, beat-cross frequency, and amplitude of lateral head displacement (ALH).
  • MTT Metabolic Activity Assay: quantified sperm metabolic activity, indicating cell viability and functionality.
  • Eosin-Nigrosin Staining: assessed sperm membrane integrity and non-viability.
  • proAKAP4 Protein Levels: used as a marker of sperm functional competence and cytoskeletal integrity.
  • Antioxidant Activity Assays (ABTS): to evaluate the free radical scavenging potential of fenugreek seed extract.

Key Findings

  • No Cytotoxicity: None of the tested fenugreek extract concentrations caused overt sperm cell toxicity under the experimental conditions.
  • Motility Changes:
    • Total sperm motility decreased over time similarly across all treatment groups, indicating no protective effect on total motility.
    • Progressive motility (important for fertility potential) and specific kinetic parameters (curvilinear velocity, beat-cross frequency, ALH) increased selectively at certain concentrations, mainly S4 (195.25 µg/mL) and S7 (24.40 µg/mL), especially at later incubation time points.
  • Metabolic Activity: Generally increased across most concentrations compared to control, particularly at early incubation times, showing enhanced sperm vitality and function soon after exposure.
  • Viability and proAKAP4 Levels:
    • The highest fenugreek concentration (S1) was associated with lower sperm non-viability (meaning better survival).
    • Higher concentrations (S1-S3) showed reduced proAKAP4 protein levels, potentially indicating some negative effect on sperm cytoskeletal functionality at very high doses.
    • Lower to intermediate concentrations (S3, S4, S6, S7) caused increased non-viability but also increased proAKAP4 levels (mainly S4, S6, S7), suggesting a complex, non-linear response depending on dose.
  • Antioxidant Properties: Fenugreek seed extract exhibited measurable antioxidant activity with significant radical scavenging capacity, which may contribute to protective effects on sperm cells by reducing oxidative stress.

Conclusions

  • Fenugreek seed extract modifies stallion sperm function in a selective, concentration- and time-dependent manner without causing overt toxicity in vitro.
  • Certain fenugreek concentrations can enhance progressive motility and metabolic activity, potentially improving sperm quality.
  • The non-monotonic effects on sperm viability and proAKAP4 protein levels highlight that both beneficial and adverse responses may occur depending on the dose.
  • Antioxidant properties of the extract likely contribute to modulation of sperm health by combating oxidative damage.
  • These findings support the potential for fenugreek extract to be used as a supplement in equine reproductive technologies but emphasize the importance of precise dosing for optimal effects.

Cite This Article

APA
Vanivska K, Kollárová I, Mezeyová I, Galovičová L, Dianová L, Halo M, Slanina T, Lenický M, Greń A, Reisenauer R, Massányi P. (2026). The effect of fenugreek (Trigonella foenum-graecum) on stallion spermatozoa motility and vitality in vitro. Vet Res Commun, 50(5), 482. https://doi.org/10.1007/s11259-026-11424-9

Publication

Veterinary research communications
ISSN: 1573-7446
NlmUniqueID: 8100520
Country: Switzerland
Language: English
Volume: 50
Issue: 5
PII: 482

Researcher Affiliations

Vanivska, Kateryna
  • Faculty of Biotechnology and Food Sciences, Institute of Applied Biology, Slovak University of Agriculture in Nitra, Trieda Andreja Hlinku 2, Nitra, 949 76, Slovak Republic. xvanivska@uniag.sk.
Kollárová, Ivana
  • Faculty of Horticulture and Landscape Engineering, Institute of Horticulture, Slovak University of Agriculture in Nitra, Trieda Andreja Hlinku 2, Nitra, 949 76, Slovak Republic.
Mezeyová, Ivana
  • Faculty of Horticulture and Landscape Engineering, Institute of Horticulture, Slovak University of Agriculture in Nitra, Trieda Andreja Hlinku 2, Nitra, 949 76, Slovak Republic.
Galovičová, Lucia
  • Faculty of Horticulture and Landscape Engineering, Institute of Horticulture, Slovak University of Agriculture in Nitra, Trieda Andreja Hlinku 2, Nitra, 949 76, Slovak Republic.
Dianová, Lucia
  • Faculty of Biotechnology and Food Sciences, Institute of Applied Biology, Slovak University of Agriculture in Nitra, Trieda Andreja Hlinku 2, Nitra, 949 76, Slovak Republic.
  • Agrobiotech Research Centre, Slovak University of Agriculture in Nitra, Trieda Andreja Hlinku 2, Nitra, 949 76, Slovak Republic.
Halo, Marko
  • Faculty of Biotechnology and Food Sciences, Institute of Applied Biology, Slovak University of Agriculture in Nitra, Trieda Andreja Hlinku 2, Nitra, 949 76, Slovak Republic.
Slanina, Tomáš
  • Faculty of Biotechnology and Food Sciences, Institute of Applied Biology, Slovak University of Agriculture in Nitra, Trieda Andreja Hlinku 2, Nitra, 949 76, Slovak Republic.
Lenický, Michal
  • Faculty of Biotechnology and Food Sciences, Institute of Applied Biology, Slovak University of Agriculture in Nitra, Trieda Andreja Hlinku 2, Nitra, 949 76, Slovak Republic.
Greń, Agnieszka
  • Faculty of Exact and Natural Sciences, Institute of Biology, University of the National Education Commission, Podchorążych 2, Krakow, 30-084, Poland.
Reisenauer, Radovan
  • Faculty of Biotechnology and Food Sciences, Institute of Applied Biology, Slovak University of Agriculture in Nitra, Trieda Andreja Hlinku 2, Nitra, 949 76, Slovak Republic.
Massányi, Peter
  • Faculty of Biotechnology and Food Sciences, Institute of Applied Biology, Slovak University of Agriculture in Nitra, Trieda Andreja Hlinku 2, Nitra, 949 76, Slovak Republic.
  • Faculty of Exact and Natural Sciences, Institute of Biology, University of the National Education Commission, Podchorążych 2, Krakow, 30-084, Poland.

MeSH Terms

  • Animals
  • Male
  • Sperm Motility / drug effects
  • Trigonella / chemistry
  • Horses / physiology
  • Spermatozoa / drug effects
  • Plant Extracts / pharmacology
  • Semen Analysis / veterinary
  • Seeds / chemistry
  • Cell Survival / drug effects

Conflict of Interest Statement

Declarations. Ethics approval: This study did not require ethical approval according to the guidelines of the Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Institute of Applied Biology. The experiments were performed in vitro using ejaculated stallion semen obtained as part of routine breeding procedures, without any direct animal experimentation. All procedures complied with institutional and national regulations for animal research and welfare. Research involving animals, their data or biological material: This study was conducted in accordance with national and institutional regulations governing the ethical use of animals in research. Ejaculated semen samples were obtained from clinically healthy breeding stallions during routine reproductive procedures, without any experimental manipulation or invasive intervention. Therefore, no ethical approval was required under the guidelines of the Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Institute of Applied Biology. All procedures complied with the principles outlined in the Basel Declaration and the International Association of Veterinary Editors’ Consensus Author Guidelines on Animal Ethics and Welfare. Competing interests: The authors declare no competing interests.

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