Abstract
Background:
Truffle extracts possess the unique properties that make them potentially valuable for medicinal purposes. The use of truffles in reproductive medicine may be of particular interest for developing medications with both spermatozoa-activating and contraceptive effects.
Aim:
Our experiment evaluated the influence of extracts of white and black truffle mushrooms on rat spermatozoa.
Settings and Design:
Animal model.
Materials and Methods:
The study utilized 28 samples of wild truffle fruiting bodies. In the experiment, we used ejaculate from male Wistar rats (n = 10, 8–12 months). Sperm samples from ten male rats were incubated at 37°C in a 96-well plate under the three conditions: control (methanol evaporated), concentrated truffle extract (25 µL), and diluted extract (1:6 ratio). After solvent evaporation, 150 µL of ejaculate was added to each well. Sixteen sperm parameters were measured using an automated analysis system after 1, 3, and 6 h of incubation.
Statistical Analysis Used:
The methods should be adequately detailed or referenced to other work. The statistical processing was performed in the Past software (V4.03) using the ANOVA analysis of variance with the Mann–Whitney test. Differences between the mean values of the parameters were considered statistically significant at P ≤ 0.05.
Results:
Research has shown that extracts from black and white truffles related to Tuber sp. have a stimulating effect on spermatozoa. Curvilinear velocity and beat cross-frequency also increased by 48% and 50%, respectively. Linearity showed a significant increase by 56% and straightness by 48%. This could be useful in the development of drugs to enhance sperm activity and lifespan. In addition, extracts from black truffles have been found to have a negative impact on spermatozoa, which could be relevant for developing new contraceptive drugs.
Conclusion:
Our study demonstrated the influence of methanol extracts of Tuber sp. mushrooms on male gametes of rats in vitro.
KEYWORDS: Biologically active compounds, infertility, rat spermatozoa, reproductive potential, Tuber sp.
INTRODUCTION
Recently, truffle mushrooms have been considered not only as an exquisite food product but also as a source of natural compounds.[1] Truffle mushrooms are rich in biologically active molecules such as ascorbic acid, phenolic compounds, ergosterols, flavonoids, terpenoids, phytosterols and polysaccharides. Due to this composition, truffles have the potential to be used as anticancer, antioxidant, antimicrobial, anti-inflammatory and anti-diabetic agents.[2] Recent studies have demonstrated that truffles have wound healing and antiviral properties.[3] Therefore, truffle mushrooms may have potential therapeutic applications.
Infertility is one of the most significant issues in modern healthcare. Approximately 13%–20% of couples worldwide suffer from infertility, regardless of race or ethnicity. One of the main causes of male infertility is poor semen quality, such as low sperm concentration or motility.[4,5]
Surgical procedures and assisted reproductive technologies are employed to attain pregnancy in cases of infertility, but their drawbacks include high costs and limited accessibility for the general population.[6,7,8] Moreover, in some countries, there are no pharmaceuticals available to restore reproductive potential.[9] Medication therapy is limited to the intake of dietary supplements,[10] which does not involve treatment for pathological conditions. Therefore, there is an urgent need for the development of safe and effective means to improve semen quality both in vitro and in vivo.
Previous studies show that one of the components of truffle mushrooms is androsthenol related to the steroid group. This molecule increases in sexual desire.[11] It is known that the alcoholic extract of desert truffle Terfezia boudieri significantly increases the levels of luteinising hormone and testosterone in rats and, consequently, acts as an aphrodisiac.[12] However, in 2014, it was found that when consuming truffle mushrooms as an aphrodisiac, attention should be paid to how truffles are prepared or cooked, their quantity and stage of ripeness, as they contain flavonoids in the form of glycosides, which act as antagonists to male sex hormones.[13] Furthermore, it is known that truffles often contain biogenic amines (serotonin, dopamine and histamine).[14]
Another study demonstrated that hydroalcoholic extract of T. boudieri exert influence on sperm and testosterone levels in male Wistar rats. The study revealed that rats injected with truffle extract experienced meaningful improvements in testosterone levels, body weight, testis weight, sperm count and sperm motility compared to the control group. These results suggest that T. boudieri may play a significant role in addressing sexual impotence and infertility in males.[15]
Truffle extracts possess unique properties that make them potentially valuable for the medicinal purposes. The use of truffles in reproductive medicine may be of particular interest for developing medications with both spermatozoa-activating and contraceptive effects. Therefore, our study aimed to investigate the effects of truffle extracts on rat spermatozoa in vitro.
MATERIALS AND METHODS
Collection of truffle mushrooms and preparation of samples
This study was conducted in accordance with institutional guidelines for the care and use of laboratory animals and followed the principles of humane treatment of animals. All experimental protocols were reviewed and approved by the Local Ethics Committee of the Faculty of Biology and Soil Science of Irkutsk State University.
The study used 28 samples of wild truffle fruiting bodies of black (Tuber macrosporum Vittad.) and white (Tuber magnatum Pico) truffles collected in Krasnodar Region from August to November 2020-2022. The mushrooms were collected near the cities of Krasnodar, Sochi and Maykop (southern part of the Russian Federation). To prevent decay, the mushrooms were placed in plastic containers with soil and rice. During transportation, the samples were kept at the temperature of 6℃–10℃. The received truffle mushrooms were cleaned with water and ethanol with a toothbrush. The mushrooms were then dried at the room temperature for 5 h to remove excess moisture and stored at −20℃. Each truffle extract was assigned a unique identification number, as presented in Table 1.
Table 1.
Numbering of extracts obtained from truffle mushrooms
| Collection location | Numbers of truffle mushroom extracts | |
|---|---|---|
| Krasnodar | Black truffles: Tuber sp. LPB2020-16, Tuber sp. LPB2020-17, Tuber sp. LPB2021-26, Tuber sp. LPB2021-27, Tuber sp. LPB2021-28, Tuber sp. LPB2021-29, Tuber sp. LPB2021-30, Tuber sp. LPB2022-39, Tuber sp. LPB2022-40, Tuber sp. LPB2022-41, Tuber sp. LPB2022-42, Tuber sp. LPB2022-44, Tuber sp. LPB2022-47, Tuber sp. LPB2022-67, Tuber sp. LPB2022-68, Tuber sp. LPB2022-69, Tuber sp. LPB2022-70, Tuber sp. LPB2022-71, Tuber sp. LPB2022-72 | |
| White truffles: Tuber sp. LPB2021-31, Tuber sp. LPB2021-32 | ||
| Sochi | Black truffles: Tuber sp. LPB2021-22, Tuber sp. LPB2021-23, Tuber sp. LPB2021-24 | |
| Maykop | Black truffles: Tuber sp. LPB2022-58, Tuber sp. LPB2022-59, Tuber sp. LPB2022-61, Tuber sp. LPB2022-63 |
Extraction of biologically active components of truffles
For extraction, 0.5 g of each truffle sample was ground into powder using a mortar and pestle, with methanol (Vecton, Russia) added in a ratio of 1:10. The obtained mixture was incubated for 1 h on a roller shaker MX-T6-S (BIOBASE, China) and then centrifuged with LC-04A centrifuge (Armed, Russia) for 10 min at 3 000 rpm. The supernatant was transferred to different tubes.[16]
Evaluation of the effect of natural compounds of truffle extracts on spermatozoa in vitro
Ten male Wistar rats aged 8–12 months participated in the experiment. The average weight of the animals was 290–350 g. They were housed in the groups of 4–5 individuals in standard plastic cages measuring 60 cm × 40 cm × 20 cm with a metal-bar lid, at a constant temperature of 25°C–27°C, humidity of 30%–70% and a 12-h light–dark cycle. The rats received a standard feeding and drinking regimen, which included compound feed and water available ad libitum.
Spermatozoa were obtained not through ejaculation but by harvesting from the proximal part of the vas deferens, which is a standard procedure for in vitro studies of sperm motility in rodents. Sperm was placed in a Petri dish with 5 ml of phosphate-buffered saline. The sperm were allowed to diffuse into the solution, after which the suspension was gently shaken at 37°C for 5 min.
The effect of truffle extracts on rat spermatozoa was assessed using a microscope with a temperature-controlled stage PLS-MY-B041A-3 (PLSUP, China), “Semen and Sperm Quality Analysis System” software (V1.12), and a 96-well plate with the optically clear bottom. The analysis was performed in the automatic mode.
The experiments were conducted at 37℃. For control conditions, 25 µL of methanol was applied to a sterile 96-well plate and dried until completely evaporated. Subsequently, 150 µL of ejaculate was added to the evaporated methanol fraction.
The experimental samples were divided into two groups. For the first group, concentrated truffle extracts were used in amount of 25 µL. For the second group, the concentrated extract was diluted in a ratio of 1:6 (2.5 µL of extract + 12.5 µL of methanol). The extracts were dried until complete evaporation of methanol (solvent) and then 150 µL of ejaculate was added. Since the methanol was completely evaporated, its effect on the spermatozoa, both in the control and experimental groups, was eliminated. The analysis was performed in three analytical replicates.
To prepare wet mount, we applied 10 µL of ejaculate onto a microscope slide and covered it with a 22 mm × 22 mm cover slip.[17,18] The microscope stage was preheated to 37℃. During the experiment, we assessed 16 physiological parameters, specifically: total of motile sperm, percent of motile sperm, fast progressive sperm, slow progressive sperm, local motile, immotile, average path velocity (VAP), curvilinear velocity (VCL), straight-line velocity, amplitude of lateral head displacement, beat cross frequency, per cent of line moving, linearity (LIN), straightness (STR), wobble and mean move angle. Physiological parameters of spermatozoa were measured after 1, 3 and 6 h of incubation at ×200 magnification. At least, 200 spermatozoa were evaluated in a total of at least five fields in each replicate, in order to achieve an acceptably low sampling error.
Statistical analysis
The methods should be adequately detailed or referenced to other work. The statistical processing was performed in the Past software (V4.03, Øyvind Hammer, Natural History Museum, University of Oslo, Oslo, Norway) using the ANOVA[19] analysis of variance with the Mann–Whitney criterion.[20] Differences between the mean values of the parameters were considered significant at P ≤ 0.05.[21]
RESULTS
The experiments revealed that after 3-h incubation, no motile spermatozoa were observed in concentrated extracts. Of the diluted extracts, eight are noteworthy for their significant effects on reproductive cells. After 3 h of incubation under the influence of diluted extract of the white truffle T. magnatum LPB2021-31, we observed a significant 56% increase in the average path sperm velocity (from 9.4 ± 2 to 14.7 ± 2.9 um/s, P = 0.01). VCL and beat cross-frequency (BCF) also increased by 48% (from 14.5 ± 3.4 to 21.5 ± 3.7 um/s, P = 0.04) and 50% (from 1.8 ± 0.6 to 2.7 ± 0.7 Hz, P = 0.04) respectively [Figure 1]. In diluted extract of the black truffle T. macrosporum LPB2022-67 after 6-h incubation, LIN showed a significant increase by 56% (from 40.7 ± 11.1 to 63.6 ± 14.8, P = 0.04) and STR by 48% (from 58.3 ± 8.8 to 86.5 ± 20.1, P = 0.01).
Figure 1.
Histogram of extract of the white truffle Tuber magnatum LPB2021-31 stimulatory effects on spermatozoa in vitro. *Statistically significant difference between groups at a significance level of P < 0.05. VAP: Average path velocity; VCL: Curvilinear velocity; BCF: Beat cross-frequency
Certain truffle extracts had adverse effects on spermatozoa, resulting in reduction of their physiological parameters when compared to the control conditions. After 3 h of incubation, we could see a significant decrease in the percentage of motile spermatozoa in extracts of black truffles: by 39% for T. macrosporum LPB2022-39 (from 96.2 ± 7.7 to 58.9 ± 3.1%, P = 0.01), 33% for T. macrosporum LPB2022-61 (from 96.2 ± 7.7 to 64.2 ± 15.1%, P = 0.01), by 24% for T. macrosporum LPB2022-63 (from 96.2 ± 7.7 to 72.8 ± 11.8%, P = 0.01) and by 22% for T. macrosporum LPB2022-72 (from 96.2 ± 7.7 to 75 ± 8.3%, P = 0.01).
After 3 h of the experiment, the local motility in black truffle extracts decreased considerably: by 51% for T. macrosporum LPB2022-61 (from 84.1 ± 17.2 to 40.1 ± 17.3 um/s, P = 0.01) [Figure 2] and by 41% for T. macrosporum LPB2022-69 (from 84.1 ± 17.2 to 34.3 ± 1.7 um/s, P = 0.01). After 6 h, the straight-line velocity in extract of black truffle T. macrosporum LPB2022-71 has also decreased by 52% (from 5.2 ± 2.1 to 2.5 ± 0.8 um/s, P = 0.04).
Figure 2.
Histogram of negative effects of black truffle extract Tuber macrosporum LPB2022-61 on spermatozoa in vitro. % motile sperm: Percent of motile sperm; WHO C: Local motile. *Statistically significant difference between groups at a significance level of P < 0.05
Thus, according to the first results of our experiments, the stimulating effect on spermatozoa in vitro was produced by extracts of black truffle T. macrosporum LPB2022-67 and white truffle T. magnatum LPB2021-31. Extracts of black truffles T. macrosporum LPB2022-61, T. macrosporum LPB2022-63, T. macrosporum LPB2022-67, T. macrosporum LPB2022-69, T. macrosporum LPB2022-71, and T. macrosporum LPB2022-72 had a negative effect on male germ cells.
DISCUSSION
Infertility is a condition of male or female reproductive system characterized by the inability to attain pregnancy after regular unprotected sexual intercourse for 12 months or longer. Male infertility is positioned to be incurable. Treatment of male infertility involves boosting immunity and taking dietary supplements (a mixture of plant extracts). The Dictionary of Natural Products database includes 55 natural compounds with spermatozoid-activating activity. The main producers of these compounds are microorganisms. Microorganisms represent a promising source of unique natural compounds for treating different human diseases. Secondary bacterial metabolites possess a variety of therapeutic activities, including antimicrobial, antiviral, antitumor, etc.[22]
In this study, we have shown that extracts of white and black Tuber sp. are primarily distinguished by their spermicidal properties. Percentage of motile spermatozoa, local motility, and straight-line velocity were significantly low. This property could be utilized to develop new contraceptives. However, two extracts from Tuber sp. purportedly showed spermatozoa-activating activity. The average path sperm velocity, VCL, BCF, LIN and STR were remarkably high.
VAP, VCL, BCF, LIN and STR are the key parameters that describe sperm motion. VAP measures the average speed of sperm along a smoothed trajectory that averages the path taken by the sperm.[23] This averaging helps to eliminate sharp changes in direction and variations in speed. VAP is used to analyse the overall ability of sperm to be mobile, as well as their potential to reach the oocyte.[24] VCL reflects the average speed of sperm along the curved path it takes.[25] This speed measurement of the actual path followed by the sperm is an important indicator of sperm activity.[23] High-VCL values often correlate with increased sperm fertilization ability, as it indicates high mobility and the ability to overcome obstacles on the way to the oocyte. BCF describes the beat frequency of the sperm’s tail. It measures how often the sperm tail crosses the average line of its movement trajectory.[26] BCF is used to assess the rhythm and synchrony of sperm tail movement. A low BCF may indicate motor defects in sperm, which in turn can affect their fertilizing capability.[27] LIN assesses how straight the sperm moves.[28] It is calculated as the ratio of the average speed along a straight trajectory (straight linear velocity [VSL]) to VCL. A high LIN indicates that the sperm is moving straight towards the oocyte. STR also measures the STR of sperm movement, but unlike LIN, STR is calculated as the ratio of VSL to VAP. This parameter helps assess how effectively the sperm follows its path. A high STR means that the sperm is moving directly towards the egg, minimising deviation from the trajectory. In procedures such as in vitro fertilisation and intracytoplasmic sperm injection, these parameters are used in selecting the most mobile and potentially viable sperm. Thus, these parameters play a major role in the diagnosis of infertility.[29]
The fruiting bodies of truffle fungi contain numerous symbionts, secondary metabolites of which exhibit various biological activities. Earlier, we demonstrated that gleba and peridium of truffle fruiting bodies have different chemical composition.[14]
Truffles have a unique composition of bacteria that have not been described in European representatives of Tuber sp.[30] Symbiotic relationships between truffles and different microorganisms can induce production of new specialised metabolites.[31] The observed biological activity of truffles can presumably be related to the synthesis of secondary metabolites by the bacterial consortium found in these fungi. This could explain unstable activity of extracts from different fruiting bodies and the limited use of truffle fungi in modern European medicine.
There is evidence indicating that anandamide plays an important role in the male reproductive system, including sperm activation. Studies have shown that the components of the endocannabinoid system involved in anandamide metabolism are present in human testicular cells. A recent study revealed that truffles contain the key endocannabinoid anandamide and enzymes responsible for its metabolism.[16] The presence of anandamide may explain the sperm-activating activity of truffle mushroom extracts.
In addition, the analysis of the problem using patents and inventions revealed several interesting references to truffle effects in Chinese medicine. Patent №CN109045095A describes a formulation that stimulates spermatogenesis, improves sperm count and viability and alleviates fatigue. The composition includes concentrated maca powder (Lepidium sp.), extract of male silkworm moth, Epimedium sp., truffle mushrooms, and raspberry.[32] Another patent №CN108740670A describes the creation of a beverage based on peptides that act as potent aphrodisiacs. One of the components of this beverage is black truffle peptide.[33] Furthermore, patent №CN112755171A describes the creation of a composition to improve male sexual function. The composition comprises three main components, one of which enhances sperm motility and improves prostate function. This component includes soy protein powder, black truffle, zinc, taurine and lycopene. Thirty healthy men aged 30–50 years consumed the above composition three times a day for a week. As a result, 83.3% of them experienced a significant improvement in sexual activity.[34] These patents provide formulations containing complex mixtures, including numerous ingredients from traditional Chinese medicine, making it difficult to study the true effects of truffle mushrooms on male reproductive system.
The invention presented in patent is based on truffle mycelium extract, which stimulates testosterone production through olfaction. The smell of truffles stimulates testosterone production in humans by activating androgen receptors in brain. Increased testosterone levels lead to improvements in spermatogenesis and ejaculate quality.[35,36] These patents have repeatedly pointed to the stimulating effects of black truffle extracts in different mixtures.
Specifically, the diluted extract of T. magnatum and T. macrosporum exhibited a stimulatory effect, indicating the presence of bioactive compounds capable of enhancing sperm motility and potentially improving male reproductive potential. In contrast, several extracts from T. macrosporum significantly reduced motility-related parameters, suggesting spermicidal activity.
These results suggest that truffle-derived compounds may serve as a promising basis for developing two distinct classes of reproductive agents:
Sperm-activating formulations aimed at treating certain forms of male infertility
Natural contraceptive agents leveraging the spermicidal properties observed in specific extracts.
In reproductive medicine, truffle-derived compounds may hold significant promise not only for enhancing sperm motility but also for supporting sperm preservation protocols used in fertility clinics. Given the observed modulation of sperm kinematic parameters, bioactive molecules from Tuber species could be incorporated into cryopreservation media to improve postthaw viability and motility of spermatozoa, which is a critical limiting factor in assisted reproductive technologies.
In addition, natural truffle extracts might serve as adjunct agents during sperm capacitation, a process essential for successful fertilisation in vitro.
Furthermore, the variability in biological activity among different truffle samples highlights the complexity of their chemical composition, likely influenced by environmental factors, symbiotic microbial communities and species-specific metabolites. This underscores the necessity for further phytochemical and pharmacological investigations to identify, isolate and standardise the active constituents responsible for these effects.
Going forward, clinical translation of these findings would require:
Standardisation of extract composition,
Pharmacokinetic and toxicological profiling,
Investigation of molecular mechanisms and signalling pathways involved in sperm modulation,
Validation of efficacy and safety in human sperm models and clinical trials. In conclusion, our study expands the understanding of the pharmacological potential of Tuber species, positioning truffle extracts as valuable candidates for both fertility enhancement and contraceptive research within the field of reproductive medicine.
CONCLUSIONS
The present study provides novel insights into the modulatory effects of methanol extracts derived from black (T. macrosporum) and white (T. magnatum) truffles on sperm motility parameters in Wistar rats in vitro. Our findings revealed a dualistic nature of truffle extracts: while certain diluted extracts significantly enhanced sperm kinematic parameters – such as VAP, VCL, LIN, STR and BCF – other extracts, particularly in concentrated form, demonstrated pronounced inhibitory effects, leading to a complete cessation of motility. In conclusion, our study expands the understanding of the pharmacological potential of Tuber species, positioning truffle extracts as valuable candidates for both fertility enhancement and contraceptive research within the field of reproductive medicine.
Author contributions
Concepts: DV, MM. Design: AA, OE. Definition of intellectual content: AY, TN, AS. Investigation: EV, NA, EI. Manuscript writing: VN, ME. Animal handling: OV, Visualisation: TY, AA.
Conflicts of interest
There are no conflicts of interest.
Data availability statement
Dataset can be made available after embargo period due to commercial restrictions on reasonable requet.
Acknowledgements
Name: Role, E.A. Pozhilenkova: assistance in conducting work with animals, A.N. Matveev: valuable advice in conducting the experiment, Giovanni Pacioni: providing valuable literary information.
Funding Statement
The study was conducted with the support of the Russian Science Foundation (project 22-76-10036).
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
Dataset can be made available after embargo period due to commercial restrictions on reasonable requet.