Effects of fenugreek ( Trigonella foenum-graecum ) on daily sperm production, epididymal transit and sperm morphology of pubertal Wistar rats

Phytotherapics are active plant materials that have played an important role in folk medicine as they favor the treatment of different diseases in many regions of the world . Different societies report improvement in the sexual function with various aphrodisiac herbal medicines, even without scientific proof. Aphrodisiacs are generally considered to be substances that increase sexual desire and/or sexual pleasure. An herbal medicine popularly used as an aphrodisiac is fenugreek. Fenugreek ( Trigonella foenum-graecum L . ) is an annual herb that belongs to the Leguminosae family. The aphrodisiac potential of fenugreek is still contradictory and not well established in the literature. Thus, the aim of this study was to evaluate the effects of fenugreek on male reproductive parameters of Wistar rats. Thirteen pubertal male rats were divided into two experimental groups (7 treated and 6 controls). Fenugreek dry extract was purchased commercially and administered daily to the treated group (3mg/animal -1 , diluted in 500 µL of water), for a period of 40 days. The control group received 500 µL of distilled water for the same period. The following parameters were analyzed: daily sperm production; sperm transit time through the epididymal duct; and sperm morphology. The results of the treated group and control group were then compared, using the non-parametric Mann-Whitney statistical test. In our study, there was no change in absolute and relative body weights of the reproductive organs and accessory glands, as well as in the sperm morphology. However, the pubertal rats treated with 3 mg/animal -1 for 40 days showed a tendency of decrease in daily sperm production and increase in epididymal transit time, suggesting a possible negative effect on male gamete production and maturation, which may lead to reduced fertility. Therefore, further studies with higher dosage and/or treatment time should be carried out to evaluate the effects of fenugreek on a complete spermatogenic cycle.


Introduction
Medicinal plants have played an important role in the treatment of different diseases in several regions of the world since ancient times, mostly for their expectorant, aphrodisiac, diuretic, diaphoretic, antispasmodic, stomachic and sedative properties (Chowdhury et al., 2018). Different societies report improvements in sexual function with different herbal aphrodisiacs even without scientific evidence (Elferink, 2000).
Currently, aphrodisiacs are generally considered to be substances that increase sexual desire (i.e. libido) and/or sexual pleasure (Hess;Boehmer, 2020), and are classified by mode of action, into three types: those that increase the libido, the potency or the sexual pleasure (Bello;Jibrin, 2015). Plant-derived aphrodisiacs are able to directly arouse male sexual libido, contribute to the maintenance of the reproductive activities, and help the neuroendocrine regulation of the mind and body in the process (Low;Tan, 2007).
Fenugreek (Trigonella foenum-graecum Linn) is an herbal medicine popularly used as an aphrodisiac. It belongs to the Fabaceae family (Gu et al., 2017) and its seeds are a source of active phytochemicals, such as trigonelline, steroidal saponins, salicylate, nicotinic acid and flavonoids, which are believed to be responsible for many of its presumed therapeutic effects considered (Basu; Srichamroen, 2010).
In Arab countries, fenugreek is used to treat wounds, diarrhea, acne, dehydration, anemia, bronchitis, rheumatism, stomach pain, high blood pressure and constipation. The seeds have important nutritional and cholesterol-lowering properties, and are traditionally used as appetite stimulants and for weight gain (Hind et al., 2017). In addition to aphrodisiac, fenugreek seeds are commonly used in the Middle East and South Asia as a spice in food preparation and also as a traditional medicine for diabetes, high cholesterol, inflammation and gastrointestinal diseases (Belguith-Hadriche et al., 2010).
The action of fenugreek on reproductive function is quite contradictory, promoting an increase in the number of morning erections and in the frequency of sexual activity (Rao et al., 2016), not causing changes in male fertility and reproductive performance (Hind et al., 2017). Thus, it is noticeable that even being used in folk medicine, the aphrodisiac potential of fenugreek is not yet well established in the literature.
Hence, to better understand the performance of fenugreek in the reproduction of male Wistar rats, it is necessary to investigate how the dry extract impacts on these animals' male reproductive parameters, since the reproductive success occurs when there are no abnormalities in sperm morphology, decrease in daily sperm production, or irregularities in epididymal sperm transit time. Thus, the general aim of this study is to evaluate the effects of fenugreek (Trigonella foenum-graecum) on daily sperm production, epididymal transit time and sperm morphology.

Animals
Thirteen pubertal male rats (55 days old), Rattus norvegicus Wistar strain were used. The animals were placed in plastic boxes measuring 40 x 32 x 17 cm (2 animals per box), under controlled conditions of light (12 h of light: 12 h of dark) and temperature (23 ± 2°C). All the animals received standardized Nuvilab diet ad libitum.

Experimental design
This study consisted of two experimental groups: animals fed with fenugreek (n = 7) and animals not fed with fenugreek or control (n = 6). T. foenum-graecum dry extract was purchased from (SM Empreendimentos Farmaceuticos LTDA) Brazil.
The treated group received fenugreek administered orally through an automatic pipette, at a dose of 3mg/animal diluted in 500 µL of distilled water, for 40 days. This dosage was calculated based on the reccomended dose for a 75 kg -1 adult male. The control group received 500µl of distilled water daily, also through automatic pipette, during the same treatment period.
Body weight records were updated weekly. The experimental protocol was approved and registered by the Ethics Committee on the Use of Animals (Comissão de Ética no Uso de Animais -CEUA) of the Federal University of Triângulo Mineiro under the number 23085.007540/2020-89.

Animal euthanasia, Daily sperm production, Epididymal transit and Sperm morphology
After the 40-day treatment, the animals were fasted for 10 h (from 10 pm to 8 am on the following day) and were only offered water during this period. The following morning, they were euthanized by supraphysiological administration of Ketamine/xylazine (150/15 mg/kg -1 ), intraperitoneally, injected in the right lower abdominal quadrant with a 22 G needle.
After confirmation of death, the right testis and epididymis were removed for the analyses of daily sperm production (DSP) and sperm transit (ST), respectively. For daily sperm production, the right testis was weighed, the tunica albuginea removed, and the parenchyma was placed in a 15ml-test tube, with 5 mL of STM solution (containing 0.9% NaCl, 0.01% thimerosal and 0.05% Triton X-100), and then homogenized. Then, 100 µL samples were removed and diluted in 900 µL of STM solution. Homogenization-resistant spermatids (stage 19 of spermatogenesis) were counted in a mirrored Neubauer chamber (Robb et al., 1978;Fernandes et al., 2007).
To analyze sperm transit time, the right epididymis was divided into two units, head-body and tail, which were weighed and homogenized according to the same protocol used to calculate DSP. Fifty µL aliquots were diluted in 950 µL of STM solution and afterwards, sperm counts were performed in a mirrored Neubauer chamber. Epididymal sperm transit time was also calculated following the methodology described by Robb et al. (1978) and adapted by Fernandes et al. (2007).
As for the evaluation of sperm morphology, the vas deferens was sectioned at the ends and washed using a syringe attached to a needle containing 1.5 mL of 10% formalin-saline solution. For the analyses, a drop of this material was placed on a histological slide and covered with a coverslip, according to the procedure described by Seed et al. (1996). A total of 200 sperms were analyzed per animal considering sperm head and tail abnormalities (Filler, 1993).

Statistical Analysis
Comparisons between the results of the treated and control groups were made using Mann-Whitney non-parametric statistical test (two-by-two comparison) (Jamovi 1.6.23). The 95% confidence interval was used (α = 0.05) (Lapponi, 1997).

Results and Discussion
The two groups did not present statistically significant differences in relation to the evolution of body weight (Table 1) over the 40 days of the experiment. Rabbits and rats submitted to diets containing 30% fenugreek seeds for 3 months did not present changes in the evolution of body weight in the course of the treatment (Kassem et al., 2006;Kamal et al., 1993). These results corroborate the present study. An increase in body weight was observed in adult rats that received aqueous extract of fenugreek (450mg kg -1 /day) for 75 days (Hind et al, 2017;Harchane et al, 2012) and in young rats receiving the following infusion for 30 days: 250 g of fenugreek seed in 1 L of water, boiled for 5 min (Rouag et al., 2020). The increase in body weight caused by T. foenum-graecum may be related to higher food intake, since it stimulates the appetite (Harchane et al., 2012).
Different factors may change the results obtained and should be taken into account when comparing the conclusions: animal age, concentration and form fenugreek was administered (dry or aqueous extract or infusion), concentration used, treatment time and others.
As for absolute and relative weights of the reproductive organs (testis and epididymis) and accessory glands (seminal vesicle and hemi-prostate), no statistically significant differences were observed between the control and treated groups ( Table 2).
The results presented by Hind et al. (2017) by feeding rats with aqueous extract of fenugreek (450mg/kg -1 /day) for 75 days corroborate those of the current study. Singh & Verma (2021) reported that adult rats receiving dry extract of fenugreek seeds (250 mg/kg -1 /day) orally, for 45 days presented a significant decrease in absolute and relative weights of testis, seminal vesicle and coagulating glands.
Daily sperm production, epididymal transit and sperm morphology of the animals treated with fenugreek were similar to those of the control animals (Table 3).
Daily sperm production is the total amount of sperm produced daily by the testicles. The number of sperm an animal is able to produce depends on the amount of functional tissue present in the testicles (Rua, 2014), if there is any damage to the testicular tissue, daily sperm production may decrease.
Rabbits fed a diet containing 30% fenugreek seed powder for 3 months presented a decrease in testis weight and sperm concentration, which indicates a negative impact on male structural and functional integrity of testicular tissues (Kassen et al., 2006).  (2022). Table 3. Daily Sperm Production -DSP (sperm x 10 6 /testis/day), epididymal transit -ET (days) and morphological changes of head and tail of the sperm of pubertal rats treated with fenugreek for 40 days and control animals.

Parameter Control (n = 6) Fenugreek (n = 7)
Testis Some studies have suggested that dry extract of fenugreek seeds promotes the increase of testosterone through different mechanisms, however, the results of these studies are not conclusive (Mansoori et al., 2020). The androgenic activity of fenugreek occurs due to the glycosides and saponins present in its seed, which may possibly act as aromatase and 5α-reductase inhibitors (Wankhede et al., 2016). The aromatase enzyme is expressed by the Sertoli cells, responsible for converting a small amount of testosterone into a highly potent estrogen, 17β-estradiol, while 5α-reductase converts testosterone into an unaromatized androgen, known as 5α-dihydrotestosterone. (Berne;Levy, 2009). Therefore, the possible increase of testosterone induced by fenugreek would be related to a higher daily sperm production, but in the present study, there was no change in the daily sperm production between the two groups.
As for epididymal transit (number of days required for the sperm to be transported by the organ), it is possible to observe an increase of 40.87% in sperm transit time through the head and body of the epididymis and 43.40% along the tail in animals treated with fenugreek. The epididymal transit time plays an important role in sperm maturation, and a change in this time may harm the process (Fernandéz et al., 2008). This faster transit of the sperm through the epididymal segments may impair the maturation process, as important events occur during this period (Robaire et al., 2006) and may hinder the fertilization process, negatively affecting the animal's fertility.
With regard to changes in sperm morphology, the average of abnormal and normal sperm was similar in both experimental groups. Studies on men who received 500 mg -1 /day of dry fenugreek extract (Furosap TM ) for 12 weeks showed a decrease in the number of abnormal sperm from the 8 th week of treatment Swaroop et al., 2017, Sankhwar et al., 2021.
Positive results concerning reproductive cells and male fertility were evidenced by Salman et al. (2021). Men undergoing treatment with fenugreek presented an increase in the amount of sperm ejaculated and a decrease in the number of cells with morphological changes.
Some results showed negative effects of the use of fenugreek on male reproductive parameters, emphasizing that dry extract of fenugreek seeds and the plant oil may produce an antifertility effect in rats (Kassem et al., 2006;Kamal et al., 1993;Ibrahim et al., 2010). The results of the study carried out by Kassem et al. (2006) showed that rabbits receiving diets containing 30% fenugreek seeds for 3 months had reduced testicular weight and sperm concentration, indicating a toxic effect of fenugreek seeds on seminiferous tubules and interstitial tissue (Leydig cells). From this perspective, the effect of fenugreek is still scientifically contradictory, varying according to dosage and treatment time.

Conclusions
Pubertal rats submitted to the treatment with dry extract of fenugreek (3 mg/animal -1 ) for 40 days did not show changes in the body weight, absolute and relative weight of the reproductive organs (testis and epididymis), accessory glands (seminal vesicle and hemi-prostate), or in sperm morphology. However, there was a tendency of decrease in daily sperm production and an increase in epididymal transit time, suggesting a possible negative effect on the production and maturation of the male gamete, which may lead to reduced fertility. Therefore, complementary studies using a higher dosage and/or treatment time should be carried on to evaluate the effects of fenugreek on a complete spermatogenic cycle (64 days), as well as the possible effects of this herbal medicine on other reproductive parameters (fertility and sexual behavior).

Acknowledgments
To the Dean of Research and Graduate Studies of the Universidade Federal do Triângulo Mineiro (PROPPG/UFTM) and to Universidade de Uberaba (UNIUBE).