Three and six grams supplementation of d-aspartic acid in resistance trained men

Geoffrey W Melville^*, Jason C Siegler and Paul WM Marshall

• * Corresponding author: Geoffrey W Melville g.melville@uws.edu.au

Author Affiliations

School of Science & Health, University of Western Sydney, Campbelltown Campus, Penrith 2751, NSW, Australia

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Journal of the International Society of Sports Nutrition 2015, 12:15  doi:10.1186/s12970-015-0078-7
The electronic version of this article is the complete one and can be found online at: http://www.jissn.com/content/12/1/15

Received:	19 November 2014
Accepted:	5 March 2015
Published:	1 April 2015

© 2015 Melville et al.; licensee BioMed Central.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

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Abstract

Background

Although abundant research has investigated the hormonal effects of d-aspartic acid in rat models, to date there is limited research on humans. Previous research has demonstrated increased total testosterone levels in sedentary men and no significant changes in hormonal levels in resistance trained men. It was hypothesised that a higher dosage may be required for experienced lifters, thus this study investigated the effects of two different dosages of d-aspartic acid on basal hormonal levels in resistance trained men and explored responsiveness to d-aspartic acid based on initial testosterone levels.

Methods

Twenty-four males, with a minimum of two years’ experience in resistance training, (age, 24.5 ± 3.2 y; training experience, 3.4 ± 1.4 y; height, 178.5 ± 6.5 cm; weight, 84.7 ± 7.2 kg; bench press 1-RM, 105.3 ± 15.2 kg) were randomised into one of three groups: 6 g.d⁻¹ plain flour (D0); 3 g.d⁻¹ of d-aspartic acid (D3); and 6 g.d⁻¹ of d-aspartic acid (D6). Participants performed a two-week washout period, training four days per week. This continued through the experimental period (14 days), with participants consuming the supplement in the morning. Serum was analysed for levels of testosterone, estradiol, sex hormone binding globulin, albumin and free testosterone was determined by calculation.

Results

D-aspartic acid supplementation revealed no main effect for group in: estradiol; sex-hormone-binding-globulin; and albumin. Total testosterone was significantly reduced in D6 (P = 0.03). Analysis of free testosterone showed that D6 was significantly reduced as compared to D0 (P = 0.005), but not significantly different to D3. Analysis did not reveal any significant differences between D3 and D0. No significant correlation between initial total testosterone levels and responsiveness to d-aspartic acid was observed (r = 0.10, P = 0.70).

Conclusions

The present study demonstrated that a daily dose of six grams of d-aspartic acid decreased levels of total testosterone and free testosterone (D6), without any concurrent change in other hormones measured. Three grams of d-aspartic acid had no significant effect on either testosterone markers. It is currently unknown what effect this reduction in testosterone will have on strength and hypertrophy gains.

Keywords:

D-aspartic acid; Resistance training; Testosterone; Estradiol; SHBG

Background

The anabolic hormone testosterone is considered to be a key determinant of training induced improvements in hypertrophy and strength. Circulating testosterone increases other anabolic hormones and directly interacts with androgen receptors and satellite cells, causing a cascade of events leading to protein synthesis and muscle growth [1],[2]. Research has previously demonstrated correlations between testosterone levels and training related strength gains [3],[4]. Furthermore exogenous elevation of testosterone to supraphysiological levels, via administration of anabolic steroids has been shown to drastically improve strength and hypertrophy [5]. Currently it is unknown whether boosting testosterone levels within normal physiological levels (mid-range to upper-range) will have a significant effect on strength and hypertrophy. Nonetheless, the supplement industry is endorsing testosterone boosters to improve training related gains. D-aspartic acid is currently recommended as a viable product to significantly raise testosterone, however research in humans only supports this recommendation in untrained men with below average testosterone levels. Moreover there is no information about the effect of different doses of d-aspartic acid on testosterone levels in humans.

Aspartic acid (C₄H₇NO₄) is an α-amino acid which is known to exist in two isoforms, l-aspartic acid and d-aspartic acid. (2R)-2-aminobutanedioic acid or d-aspartic acid (DAA), previously believed to be exclusive to brain tissue in octopus, squid and cuttlefish, has more recently been shown to exist in mammals [6]. Free DAA is found in tissues and cells related to the central nervous and endocrine systems [7],[8]. DAA is believed to stimulate the production and release of testosterone through multiple pathways of the hypothalamic-pituitary-gonadal (HPG) axis. It has been shown to increase steroidogenic acute regulatory protein (StAR) gene expression in rat Leydig cells [9]. StAR is a key regulator for the transport of cholesterol from outside the mitochondrial membrane to the inner membrane [7]. By increasing levels of StAR DAA may indirectly increase testosterone, as the transportation of cholesterol is believed to be the rate limiting step in the production of testosterone [7]. In vitro rats studies demonstrated that DAA increased levels of testosterone, luteinizing hormone, progesterone [6] and growth hormone [10]. This is believed to occur due to the accumulation of DAA in the anterior pituitary and testes [10]. Additional in vitro studies on isolated rat testes [6] and Leydig cells [11] indicate that DAA increased the rate of testosterone synthesis in a dose dependent manner. In these animals the maximal effective dose of DAA, which elicited the greatest hormonal response (LH, testosterone and progesterone), was 1 μmol.g⁻¹[6]. In humans the effects of different dosages of DAA on basal testosterone is unclear.

To date only two studies on DAA supplementation have been conducted on humans. Topo et al. [12] demonstrated that after 12 days of supplementation (3.12 g.d⁻¹), levels of testosterone were significantly increased by 42% (4.5–6.4 ng.ml⁻¹). They recruited a cohort of healthy sedentary male IVF patients (27–37 years), with low initial testosterone levels (~4.55 ng.ml⁻¹). Contrastingly Willoughby and Leutholtz, reported that after 29 days of supplementation (3 g.d⁻¹) and resistance training, levels of total testosterone and free testosterone were not significantly altered. In this study resistance trained men (age: 22.8 ± 4.67 years old; training age: > 1 year) were recruited and this cohort exhibited higher initial testosterone levels (~7.96 ng.ml⁻¹) [13]. The difference in outcome between these two studies may in part be explained by training status and accompanying basal testosterone levels. Basal testosterone levels of RT men range from approximately 5.8–8.6 ng.ml⁻¹ (20–30 nmol.l⁻¹), [4],[14] and untrained men range from about 4.9–6.6 ng.ml⁻¹ (17–23 nmol.l⁻¹) [15]-[17]. Furthermore current research has only explored one dose response of DAA, 3 g.d⁻¹[12],[13], hence the maximum effective dose for humans is yet to be determined.

Supplement companies are currently recommending three grams of DAA once to twice a day, and these recommendations have been drawn from the only dosage studied in humans (3 g.d⁻¹). It is reasonable to believe that in RT males, a higher dose may be required to further increase testosterone levels. As such the primary aim of this study was to evaluate the effects of two doses of d-aspartic acid (3 g and 6 g) on basal testosterone levels in resistance trained men. A secondary aim was to establish if a relationship exists between initial testosterone levels and responsiveness to DAA. It was hypothesised that; (a) testosterone levels would be unchanged in the 3 g group; (b) testosterone levels would be increased in the 6 g group; and (c) lower initial testosterone levels would correspond with an increased responsiveness to DAA.

Methods

Subjects

The institutional review board approved the study and participants provided written informed consent prior to testing and participation. A total of twenty-four participants from the local area completed this study (Table 1). To be eligible participants had to be: male; aged 18–36; have no acute or chronic medical conditions; have the ability to bench press 100% bodyweight; and had been performing regular resistance training exercise for at least three days per week for the previous two years. None of the participants were supplementing their diet with any ergogenic or testosterone booting supplements prior to testing. All participants provided written consent and completed a medical history check. The study was approved by the University of Western Sydney human research ethics committee, and carried out in accordance with the declaration of Helsinki.

Table 1. Participant demographics

Experimental approach to the problem

This was a randomised, double-blinded, and placebo-controlled design to examine the effects of d-aspartic acid supplementation on basal testosterone levels following a two week supplementation protocol. Participants were assigned to one of three experimental groups: placebo (D0), three grams of DAA (D3) and six grams of DAA (D6). All participants consumed 10 opaque capsules each morning with breakfast for two weeks. They contained either: six grams of flour (D0, n = 8); a mixture of three grams each of flour and DAA (D3, n = 8); or six grams of DAA (D6, n = 8). Participants were randomly allocated to treatment groups following a block randomisation procedure based on a computer-generated list of random numbers. Placebo, mixed and supplement were provided in identical opaque capsules to improve blinding. Group allocation was managed by a technical officer, whilst investigators were kept blind to group assignment throughout the intervention. All participants followed an upper/lower body split resistance training program for a full month, with the initial two weeks of training (washout period) performed without supplementation (Figure 1). Three timepoints were used to obtain testing data: T1, T2 and T3 (Figure 1).

Figure 1. Timeline of the study. After completion of T1, subjects began training four days per week. Daily supplementation commenced after T2 (). T1-3 included fasted blood draws ().

Experimental procedures

Testing sessions consisted of a fasted blood draw, then 1-RM bench press evaluation. Initial baseline blood measures were taken at two timepoints (T1 & T2) and averaged to ensure accuracy in baseline assessment of these markers (Figure 1). After T1 prescribed training commenced for four weeks. After testing session T2 daily supplementation begun with training continuing as before. Post-measures were taken after these last two weeks of training and supplementation, at the end of week 4 (Figure 1). The supplemental period of two weeks was chosen as this has been previously shown to be a sufficient time period to see a change in total testosterone levels [12].

1-RM testing

Bench press dynamic strength one repetition max (1-RM) was measured before the standardisation period (T1), beginning of experimental period (T2) and post experiment period (T3) (Figure 1), as part of eligibility testing. Correct form included depth to the level of the chest, with feet not leaving the floor, and the backside not leaving the bench at any point during the repetition. The protocol for 1-RM testing involved one warm up set of 10 reps at approximately 50% of their estimated 1-RM, followed by two more warm ups at approximately 70% and 80% with only 1–2 reps. After the warm ups participants attempted 1-RMs with incrementally increasing weight. The weight achieved prior to the failed attempt was recorded as the 1-RM. A participant’s 1-RM was achieved within five attempts and adequate rest between attempts was adhered to (3–5 mins) [18].

Fasted blood draws

All blood draws were obtained via venepuncture of the antecubital vein after a 12 hour fast. Participants were also instructed to avoid strenuous exercise and alcohol consumption the day before the draw. Blood draws were conducted by a trained phlebotomist and subsequent draws were planned for the same time of morning (7:00–10:00 am) for each particular participant, to prevent any effect of diurnal variation. Whole blood was collected using serum separator tubes (SST™ II Advance, BD Vacutainer®). They were then allowed to clot for 45 minutes and centrifuged using a fixed angle rotor centrifuge: ADAMS® Compact II Centrifuge, V:227 (Becton Dickinson & Co) (828 × g, at 2700 rpm) for 15 minutes in an air conditioned room (19°C). Serum was aliquoted and stored at −80°C until analysis (Douglas Hanly Moir Pathology, Macquarie Park, NSW, Australia). Single analysis of serum was conducted for total testosterone, estradiol, sex-hormone-binding-globulin (SHBG) and albumin. Testosterone and SHBG was measured via electrochemiluminescent (ECL) immunoassay, on a Roche E170 system (Roche Diagnostics). Albumin was measured via bromocresol green (BCG) succinate buffer method, on an Abbott 16000. Estradiol was measured via chemiluminescent microparticle immunoassay on an Abbott i2000. Free testosterone was calculated from total testosterone, SHBG and albumin.

Training standardisation

Participants trained for four days per week over a one month period. The prescribed training for each exercise consisted of four sets of a repetition maximum range of 8–10. If the repetition range wasn’t met, participants were asked to lower or raise the weight in the next session. Exercises during the upper body session were: barbell bench press; overhand pulldown; barbell overhead press and underhand pulldown. The lower body session consisted of: back squat; good morning; leg extensions; and straight leg calf raises. Adherence was monitored via training diaries and supervised sessions (minimum 1 × per week).

Dietary intake

Participants were asked to control their diet, by avoiding any major changes throughout the study duration. To monitor their diet they were asked to weigh and recorded their food intake for three days each of the first and last week; two training days and one non-training day. These three days were averaged to get a daily mean for week one and four. The food diaries were entered into CalorieKing (Australian Edition 4.0), then analysed for caloric and macronutrient daily intakes (protein, carbohydrates and fats) and normalised to bodyweight.

Statistical analysis

Analyses were conducted using IBM SPSS Statistics for Windows version 21.0 (Armonk, NY: IBM Corp), and the level of significance was set at P < 0.05. Data are shown as mean ± S.D. The distribution was tested for normality using the Kolmogorov-Smirnov test. Paired sample statistics were run on total testosterone (TT), free testosterone (FT), estradiol (E₂), sex-hormone-binding-globulin (SHBG), and albumin (ALB) to determine the stability of these blood measures over the standardisation period. As these measures were found to be unchanged they were each computed (averaged) into one baseline measure. Univariate analysis of the absolute change scores: Δ=(T3−T1+T22)was conducted, with the baseline scores: PRE=(T1+T22)as covariates (Figure 1). Pairwise comparisons with Bonferroni correction were performed if a group effect was observed. To explore the responsiveness of the supplement, linear regression analysis was conducted on the baseline and change scores of TT and FT, of the experimental groups (n = 16).

Results

Analysis of the POST values revealed no main effect for group with E₂ (P = 0.47), SHBG (P = 0.07) and ALB (P = 0.32). Post values of D6 TT were significantly reduced (~12.5%) as compared to the pre values (P = 0.03; 5.9 to 5.1 ng.ml⁻¹). FT in group D6 was significantly decreased (429.1 to 363.4 pmol.l⁻¹) as compared to D0 (439.6 to 480.9 pmol.l⁻¹) (P = 0.005) but not D3 (534.9 to 524.3 pmol.l⁻¹) (P = 0.06) (Figure 2). Diet analysis revealed no significant changes in macronutrient (CHO: P = 0.74; PRO: P = 0.99; FAT: P = 0.54) and caloric intakes (P = 0.64) during the study. Regression analysis revealed no significant correlation between baseline total testosterone levels and total testosterone change (r = 0.10, P = 0.70), and no significant correlation between baseline free testosterone and free testosterone change (r = 0.32, P = 0.23).

Figure 2. The absolute change of free testosterone. *statistically significant (P < 0.05).

Discussion

The primary findings of the current study were, 1) resistance trained men consuming six grams of d-aspartic acid daily demonstrated significant reductions in total and free testosterone after 14 days of d-aspartic acid supplementation, and 2) the responsiveness to d-aspartic acid supplementation was unaffected by initial testosterone levels (total or free) in resistance trained men.

Our results demonstrate that in resistance trained men three grams daily of d-aspartic acid had no significant effect on total testosterone, estradiol, sex-hormone-binding-globulin, and albumin. This is contrary to the evidence provided by Topo et al. [12], where the cohort consumed the same dose over 12 days and reported elevated total testosterone levels (~42%). Baseline testosterone levels of the current study were higher than values found in Topo et al. [12] (6.3 and 4.5 ng.ml⁻¹ respectively), presumably because the cohort in the Topo et al. study were sedentary [12]. In resistance training literature, total testosterone levels range from 5.8–8.6 ng.ml⁻¹[4],[14] for trained individuals and 4.9–6.6 ng.ml⁻¹ for untrained [15]-[17]. The increase in testosterone observed in Topo et al. [12] was likely due to the fact that testosterone levels were low enough for d-aspartic acid to have an effect. In comparison our results in the D3 group mirror the results seen in the study by Willoughby & Leutholtz [13], where the total testosterone levels fall within levels observed in resistance trained males [4],[14].

It was observed in the six gram group that total testosterone was significantly reduced from baseline by ~12.5%, with a parallel decrease in free testosterone ~15.3% (see Table 2). Previous research has demonstrated that in resistance trained men, free testosterone can increase due to training [19]. A reduction in calculated free testosterone in this study is due to a reduction in total testosterone, an increase in the binding proteins or a combination of the two occurring. Within the context of increasing total testosterone a maximum effective dosage (MED) is observed in rat studies [6]. At the higher dosages there were significantly increased accumulation of d-aspartic acid observed in the pituitary and testes [6]. A dose response increase in total testosterone was observed until 1 μmol.g⁻¹. Each increase in dose past 1 μmol.g⁻¹ the rise in testosterone was reduced [6]. It could be theorised that 6 g.d⁻¹ may be affecting negative feedback mechanisms of the HPG axis, thus reducing pituitary initiated production of luteinizing hormone and in turn testosterone levels. Furthermore d-aspartic acid could also be over-accumulating within the testes. This may be creating a disruptive effect on the mobilisation of cholesterol from the outer membrane to the inner [7], which would attenuate testosterone production. As this was the first study to administer a six gram dosage of d-aspartic acid, these mechanisms can only be speculated due to the lack of data available on the utilisation of d-aspartic acid in humans.

Table 2. PRE (Baseline), POST (T3), and Change Scores (Δ) of hormonal markers

The reductions in testosterone observed in this study are important to consider, owing to the negative impact it could have on training gains within this population. Resistance trained men have higher levels of strength and hypertrophy compared to novice trainers and also exhibit higher basal testosterone levels [4],[13]-[17], which suggest a link between basal total testosterone levels and training related gains. A decrease in total testosterone with a concurrent decrease in free testosterone could reduce the likelihood of interaction with androgen receptors in muscles and nerves, which would reduce the speed of testosterone initiated muscle protein synthesis [1]. Over time this could translate into reduced training gains. Conversely, alterations of testosterone within normal physiological ranges may not be clinically significant. Research indicates that when total testosterone levels are observed outside of normal healthy ranges (4.9-8.6 ng.ml⁻¹) it affects strength and hypertrophy. In the case of hypogonadism where testosterone levels are low this negatively affects strength and hypertrophy, and with the use of steroids a positive affect is seen [5],[20]. The changes observed in the current study reflect minor alterations with respect to normal physiological ranges. It is currently unknown if these fluctuations are detrimental to training gains.

A potential limitation of this research may be the study length. The short term nature of a two week supplementation period will answer only acute hypotheses. The observed reduction in testosterone may rebound, or even decrease further and a longer term training study would be able to better explain the effects of this supplement. Moreover it would be able to delineate changes in strength and or hypertrophy, and observe whether d-aspartic acid affects training related gains positively or negatively.

Conclusion

Many testosterone boosting supplements are commercially available without sufficient research to support their efficacy. The present study has demonstrated that 3 g.d⁻¹ of d-aspartic acid was inadequate to affect any hormonal markers and that 6 g.d⁻¹ significantly reduced total testosterone and free testosterone levels, with no concurrent change in other hormones tested. It is currently unknown if any negative consequences of this reduction, with respect to strength and hypertrophy will occur over time. The need for longer-duration research utilising six grams of d-aspartic acid is clear. Future research should explore supplementation of 6 g.d⁻¹ over a longer period and observe any correlations between basal testosterone levels and changes in hypertrophy and strength.

Competing interests

The d-aspartic acid supplement used in this study was commercially sourced. The authors have no undisclosed professional relationships with companies or manufacturers that would benefit from the results of the present study. The authors declare that they have no competing interests.

Authors’ contributions

GM, PM and JS contributed to the study conception and design, GM acquired the data, performed data analysis and interpreted the data; all authors were involved in drafting the manuscript and have given final approval of the published version.

Acknowledgments

The author’s thank the volunteers who participated in the study.

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The inhibitory effects on adult male reproductive functions of crude garlic (Allium sativum) feeding

The inhibitory effects on adult male reproductive functions of crude garlic (Allium sativum) feeding
Imen Hammami^{1, 2}, Afef Nahdi¹, Claire Mauduit^{2, 3}, Mohamed Benahmed², Mohamed Amri⁴, Awatef Ben Amar⁵, Semy Zekri⁵, Ahmed El May⁶, Michele Veronique El May¹

¹Research unity nº 01/UR/08-07, Faculty of Medicine, Tunis 1007, Tunisia
²Inserm, U407, Oullins, F-69921, France; University of Lyon, Oullins F-69921, France
³Civil Hospitals of Lyon, Hospital Center of Lyon-Sud, Laboratory of Anatomy and Cytology-Pathology, Pierre-Benite
cedex F-69495, France
⁴Nutrition Physiology Laboratory, Faculty of Sciences, Tunis 1006, Tunisia
⁵Laboratory of Electronic Microscopy, Faculty of Medicine, Tunis 1007, Tunisia
⁶Laboratory of Immuno-histo-cytology, Salah Azaiez Institute, Tunis 1006, Tunisia

Abstract

Aim: To investigate the effects of crude garlic on adult male rat reproductive functions.  Methods: Thirty male rats were divided into five groups: group 1 (untreated) and groups 2, 3, 4 and 5 were fed for 30 days with 5%, 10%, 15% and 30% crude garlic, respectively. Testes and accessory organs were weighed and some markers were assessed. Light and electron microscopy observations were also performed.  Results: A significant decrease was observed in the body weight of groups 4 (14%; P < 0.01) and 5 (20%; P < 0.01); of the prostate weight in group 5 (29.1%; P < 0.05) and of seminal vesicle weight in groups 3 (14.4%; P < 0.01), 4 (18.3%; P < 0.01) and 5 (27.3%; P < 0.01). In contrast, testis and epididymis weights were unchanged. In epididymis tissue, the alpha glucosidase activity and the spermatozoa density were unchanged. The treatment resulted in a significant decrease in testosterone serum levels in groups 3 (77.3%; P < 0.01), 4 (77.3%; P < 0.01) and 5 (90.9%; P < 0.01), associated with a significant increase in LH serum levels (P < 0.01). Testicular histology showed a dose-dependent increase in the percentage of empty seminiferous tubules. Moreover, testicular function was affected; a significant decrease in phosphatase acid activity (P < 0.01) and testosterone (P < 0.05) contents were observed. Conclusion: Crude garlic consumption during 1 month reduced testosterone secretion and altered spermatogenesis at 10%, 15% and 30% doses. (Asian J Androl 2008 Jul; 10: 593_601)

Keywords: crude garlic; spermatogenesis; testosterone; luteinizing hormone; testis; sexual accessory organs; Sertoli cell; Leydig cell; germ

Correspondence to: Dr Claire Mauduit, INSERM U407, Faculty of Medicine Lyon-Sud, B.P 12, Oullins Cedex 69921, France.
Tel: +33-42623-5924 Fax: +33-42623-5916
E-mail: mauduit@sante.univ-lyon1.fr
Received 2007-04-17     Accepted 2007-08-23
DOI: 10.1111/j.1745-7262.2008.00358.x

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1 Introduction

Alternative medicines, automedication and phytotherapy are part of the way of life of most populations, particularly in Africa. Side effects of many medicinal plants on fertility are unknown. Some of these plants contain estrogenic substances and, therefore, might alter gametogenesis production [1]. Some plants like Morinda lucida, Ricinus communis and Yohimbe are known to cause reductions in sperm density [2], to alter androgenic secretion [3] and to reduce mobility and density of mice spermatozoa [4].

Allium sativum (As) is a frequently used plant in Mediterranean cooking. In Tunisia, it is regularly consumed at various doses both crude and cooked. Therapeutic virtues of this plant are numerous [5]; however, its impacts on the male reproductive system have been not clearly defined. Some studies have reported that garlic impairs testicular function [6] and has spermicidal effects on spermatozoa [7, 8], but others demonstrate its beneficial effects on recovery of testicular functions [9]. These discrepancies could be related to the type of preparations, doses and way of administration.

In our study, we investigated the effects of chronic consumption of crude garlic, as is largely used in Mediterranean cooking, on the following variables of male rats' reproductive functions: testicular and plasma testosterone, luteinizing hormone (LH) levels, prostate and seminal vesicle markers, sperm density and testicular integrity on histological sections.

2 Materials and methods

2.1 Plant and preparation

The type of As used in the present study was "spring garlic". This variety has pink bulbs and is planted between December and March (according to the weather) in Tunisia and collected in July. This type of garlic contains 2.1% proteins, 30% carbohydrates, 1.5% fibre, 0.2% fat, 0.015% vitamins and 0.7% minerals. The plant (As) used in this study was grown in Tunisia and purchased at a local market. Every day the garlic pellets were made by mixing peeled cloves of garlic with powdered standard rat pellet diet (Industrial society of food, Sfax, Tunisia) at four doses: 5%, 10%, 15% and 30%. For example, the 30% pellets for one rat were prepared by mixing 9 g of crude garlic with 21 g of powdered standard diet in 5 mL of water. Cloves were crushed in distilled water to minimize volatile compound loss. A similar volume of water was added to the other doses.

2.2 Animals and treatment

A number of 30 adult male Wistar rats (Pasteur Institute of Tunis, Tunisia), whose average weight ranged between 200 g and 250 g, were used for the study. The animals were housed with proper aeration at 25 ± 2°C, and were given tap water ad libitum. The rats were allowed to acclimatize in the laboratory for a period of 1 week before the beginning of the study. The rats were randomly assigned into the different groups (of six animals each) using a hazard permutation table. Control animals received a standard pellet diet (group 1). The other groups received a diet supplemented with 5%, 10%, 15% and 30% of As (for groups 2, 3, 4 and 5, respectively). Every day, 30 g of food (garlic mixed with standard diet) was given to each rat. The animals consumed 30 g of food daily, as no pellet was observed the following day. All rats were weighed daily. After 30 days of treatment rats were killed and a cardiac blood sample was taken from each rat and then put into a sterile tube. Blood was allowed to clot at room temperature. When the clot was retracted, the sample was centrifuged at 3 000 × g for 15 min and the serum was transferred to a new tube. The serum samples were stored frozen at _20ºC until use. All the rats were killed by decapitation the same day between 09:00 and 11:00 o'clock. Reproductive organs were dissected out and weighed.

All studies on animals were conducted in accordance with current regulations and standards approved by the Faculty of Medicine of Tunis animal care committee.

2.3 Hormonal analysis

The same radioimmunoassay (RIA) system was used to measure testosterone contents in both testicular tissue and serum samples. The RIA kit was obtained from Biosource (Nivelles, Belgium). The intra-assay and interassay coefficients of variations (CV) were 4.6% and 6.2%, respectively. The detection limit of the testosterone assay was 0.05 ng/mL. LH (Biocode-Hycel, Liège, Belgium) concentrations were determined according to the manufacturer's recommendations. The detection limit of the LH assay was 0.05 ng/mL. The intra-assay and interassay CV were 8.2% and 6.8%, respectively.

2.4 Tissue biochemistry

2.4.1 Testis

Testosterone and cholesterol contents were determined in testicular tissue. One testis was crushed into 2 mL of 0.9% NaCl in distilled water. The homogenate was centrifuged at 13 000 × g for 10 min. The supernatant was removed and used for determination of testosterone and cholesterol contents with the same assay as for blood samples. The results were expressed as mg/g or ng/g of testicular tissue for cholesterol and testosterone, respectively. Cholesterol levels (Diagnostics Elitech, Sees, France) were assayed with a colorimetric method [10]. The intra-assay and inter-assay CV were 1.7% and 3.8%, respectively. The detection limit was 0.05 g/L.

Acid phosphatase activity was determined using a colorimetric assay (Diagnostics Elitech, Sees, France) according to the manufacturer's recommendations. The intra-assay and interassay CV were 1.6% and 2.3%, respectively. The detection limit of acid phosphatase activity assay was 0.5 µmol/min/L. 0.5 g of testicular tissue were homogenized in 2 mL of citric acid buffer (0.1 mol/L citric acid, 0.2 mol/L Na₂HPO₄, pH 6.2, supplemented with 0.4% Triton X-100 solution) and centrifuged at 80 000 × g at 4ºC for 30 min. The reaction medium containing 0.1 mL supernatant, 0.05 mL 4-paranitrophenol (PNP, 23 mmol/L) and 0.5 mL buffer (0.1 mol/L citric acid, 0.2 mol/L Na₂HPO₄, pH 5.0) was incubated at 37ºC for 30 min. Then, 2.5 mL of NaOH (0.2 mol/L) was added to stop the reaction, and the absorbance (Metertek SP-850, Metertech, Taipei, Taiwan) was recorded at 405 nm. A standard PNP curve was obtained using the same method. Acid phosphatase activity was expressed as µmol/min/g of tissue.

2.4.2 Epididymis

One caudal epididymis of each rat was cut, homogenized in citric acid buffer (0.1 mol/L citric acid, 0.2 mol/L Na₂HPO₄, pH 6.2, supplemented with 0.4% Triton X-100 solution) and centrifuged at 80 000 × g at 4ºC for 30 min. The alpha-glucosidase activity was measured using the colorimetric method [11]. The reaction system contained 1.2 mL buffer (69 mmol/L citric acid, pH 6.8), 0.2 mL paranitrophosphateglycerol (PNPG, 23 mmol/L) and 0.2 mL supernatant. The reaction medium was incubated at 37ºC for 4 h and 0.25 mL Na₂CO₃ (0.1 mol/L) was added to stop the reaction. The absorbance was measured at 400 nm with a Metertek SP-850 (Metertech, Taipei, Taiwan) spectrophotometer and PNPG content was estimated to a standard curve. The alpha-glucosidase activity was expressed as µmol/min/g of tissue. The detection limit of alpha-glucosidase activity assay was 0.5 µmol/min/L. The intra-assay and interassay CV were 2.1% and 2.6%, respectively.

2.4.3 Prostate and seminal vesicle

Extraction procedures were similar for prostate and seminal vesicle. 0.2 g of tissues were homogenized in 2 mL of 0.33% perchloric acid at 4ºC and centrifuged at 2 500 × g for 10 min. Then, 1 mL of the supernatant was added to 0.5 mL K₂CO₃ (0.75 mol/L). The reaction medium was centrifuged at 2 500 × g for 10 min and supernatants were used for determination of prostate citric acid (r-Biopharm, Darmstadt, Germany) and seminal vesicle fructose (r-Biopharm, Darmstadt, Germany) using an ultra violet method according to the manufacturer's recommendations. The detection limits were 0.5 mg/L and 0.4 mg/L, respectively. The interassay CV were 4.2% and 1.8% for prostate citric acid and seminal vesicle fructose, respectively. The intra-assay CV were 1.3% for prostate citric acid and 1.8% for seminal vesicle fructose.

2.5 Sperm density

The caudal epididymis was removed, and cut in small pieces into 1 mL of 0.9% NaCl. The NaCl solution was transferred into a new tube. The epididymis tissue was rinsed with 0.5 mL of NaCl that was added to the previous tube. The NaCl solution containing spermatozoa was incubated for 30 min at room temperature. Then, to 50 µL of the spermatozoa suspension was added 200 µL of formaldehyde 1%. The number of spermatozoa was determined using a Thomas' cytometer cell. The results were expressed as the number of spermatozoa (10⁶/mL).

2.6 Histopathological studies

Testes were fixed in a 10% formaldehyde solution, passed through ascending series of ethanol baths, cleared in toluene and embedded in paraffin. Tissues were sectioned at 4 µm and stained with haematoxylin and eosin. For the determination of the number of empty seminiferous tubules, a slide from each animal was used. All the seminiferous tubules were counted and the results were presented as a percentage of empty seminiferous tubules. For the determination of the seminiferous tubule area, only round and almost round (oval-shaped) tubules were analyzed. To calculate the area, the diameter was measured (with a micrometer objective) for round seminiferous tubules and the small and large diameters were measured for oval-shaped tubules. Some fragments of testis were processed for electron microscopy. They were fixed in 4% glutaraldehyde, postfixed in a 1% osmium tetroxide solution, and embedded in Epon 812. Ultra-thin sections were observed on a JEOL1010 transmission electron microscope after lead citrate and uranyl acetate contrast.

2.7 Statistical analysis

All data are presented as mean ± SD and median. Statistical analyses were performed using SPSS 10.0 for Windows (SPSS, Chicago, IL, USA). To determine whether there were differences between all groups the Kruskall-Wallis test was performed and this was followed by the Mann-Whitney U-test to determine the significance (P < 0.05) of the differences between the pair of groups.

3 Results

3.1 Body and organ weights

Compared to the control group, rats in groups 4 and 5 showed significant decreases in body weight, 14% (P < 0.01) and 20% (P < 0.01), respectively (Table 1). Concerning the weight of the reproductive organs, crude garlic treatment significantly decreased seminal vesicle weight in group 3 (14.4%; P < 0.01), 4 (18.3%; P < 0.01) and 5 (27.3%; P < 0.01). The prostate weight (Table 1) was significantly decreased (by 29.1%; P < 0.05) only in group 5. In contrast, no significant modification of testis and epididymis weights was observed after crude garlic treatment.

3.2 Hormonal measurement

A significant decrease in serum testosterone levels was observed in groups 3 (77.3%; P < 0.01), 4 (77.3%; P < 0.01) and 5 (90.9%; P < 0.01), accompanied by significant increases in LH concentration (P < 0.01) at these doses (Table 2).

3.3 Accessory gland functions

The treated rats showed no significant reductions in alpha-glucosidase activity in caudal epididymis and no significant reductions in spermatozoa density in caudal epididymis (Table 3).

However, prostate citric acid content was significantly decreased (19.4%; P < 0.05) in group 5. There were 32.7%, 63.8% and 75.1% reduction in seminal vesicle fructose at 10%, 15% and 30% doses of As, respectively (P < 0.01) in comparison to the untreated rats (Table 3).

3.4 Testis

3.4.1 Testicular morphology and ultrastructure

Morphological alterations of seminiferous tubules were observed in group 5 (30% of As) (Figure 1C and 1D) when compared to the control testis (Figure 1A and 1B). A significant and dose-dependent increase in the percentage of empty seminiferous tubules was observed after treatment with 10% (P = 0.002), 15% (P = 0.002) and 30% (P = 0.004) of crude garlic (Table 4). An approximate threefold increase was observed in the percentage of empty seminiferous tubules in the group fed 30% garlic as compared to the control group. In contrast, the area of the seminiferous tubules was unchanged by As feeding (63.43 ± 3.05 µm² for 30% As vs. 63.08 ± 2.66 µm² for the control group). The testicular ultrastructure of rats treated during for 30 days with 30% of As displayed cellular alterations (Figure 2). Sertoli cells had a reduced volume and presented vacuolization, sparse organelles and a few scattered mitochondria in their cytoplasm (Figure 2D) compared to untreated rats (Figure 2A). Nuclear degeneration was evident in the primary spermatocytes and spermatids: nuclear envelopes were frequently interrupted (Figure 2E). Leydig cells displayed more lipid droplets (Figure 2F) than untreated ones (Figure 2C).

3.4.2 Testicular functions

In the testicular tissue, the acid phosphatase activity was significantly decreased in groups 2 (48.3%; P < 0.01), 3 (47.4%; P < 0.01), 4 (33.2%; P < 0.01) and 5 (48.1%; P < 0.01). Similarly, a significant decrease in intra-testicular testosterone concentration was observed in groups 2 (33.3% decrease; P < 0.05), 3 (73.3% decrease; P < 0.05), 4 (80% decrease; P < 0.05) and 5 (89.3%; P < 0.05). In contrast, no significant change in intra-testicular cholesterol concentration was detected compared to the values observed in the control (Table 4).

4 Discussion

In the present study, rats fed a diet consisting of 15% or 30% crude garlic had significantly reduced body weights compared to rats who did not consume garlic. Our results are in accordance with the study by Dixit and Joshi [6] reporting a decrease in body weight after treatment with a powder garlic preparation by daily gavage. Concerning the accessory gland functions, the present study showed no significant difference between treated and untreated groups in epididymis α-glucosidase activity or in sperm density in caudal epididymis. However, we detected significant increase in the number of empty seminiferous tubules in the testes from rats fed 10%, 15% or 30% As. One possible explanation for the absence of significant modifications in sperm density observed in caudal epididymis in the present study might be that the treatment was conducted during 30 days, whereas the rat seminal cycle lasts 53 days. Al-Bekairi et al. [12] reported an increase in epididymis spermatozoa after feeding rats with garlic water extract over 3 months. The reasons for this discrepancy in the studies could be linked to the difference in the garlic preparation (crude garlic versus water extract).

We showed here that a reduction in prostate weight was associated with a decrease in citric acid content when rats were fed 30% garlic. These results suggest a dysfunction of the prostate gland, which might be a result of low testosterone levels, because the secretion of citric acid is regulated by androgens [13]. Moreover, a low fructose concentration and a reduction in seminal vesicle weight were observed in rats treated with high doses of garlic. These results could also be attributed to decreased testosterone levels. Fructose provides energy for sperm motility [14]: an interesting question to address would be whether sperm motility is modified in rats fed crude garlic.

In the testis, acid phosphatase is widely distributed in lysosomes of Sertoli cells, spermatogonia and late spermatids [15]. Activities of free lysosomal enzymes have been shown to rise when testicular steroidogenesis is increased [16]. In the present study, the decrease in acid phosphatase activity might reflect decreased testicular function in the treated rats and might be associated with the reduced secretion of testosterone. However, the possibility exists that the effects observed here on male reproductive functions were linked to the body weight loss detected at doses of 15% and 30% As. However, hallmarks of the negative effects of As on male reproductive functions (such as decreased seminal vesicle weight and plasma and testicular tissue testosterone contents) were also observed at doses of 5% and 10% As that did not induce a body weight loss. Moreover, by using a protocol of daily gavage administration, that reduced the possibility of adulteration of rats pellets, Dixit and Joshi [6] showed that As induced a reduction in accessory gland weight and hypospermatogenesis.

Administration of crude garlic resulted in decreased serum and testicular testosterone levels, suggesting that crude garlic has an inhibitory effect on testosterone production. Interestingly, this effect is dose-dependent (10%, 15% and 30%). The reduction in circulating and intra-testicular testosterone levels was associated with elevated LH levels in rats treated with 10%, 15% and 30% crude garlic. These results suggest a diminished responsiveness of Leydig cells to LH and/or a direct inhibition of testicular steroidogenesis and as such a testicular alteration in the gonadotropin-testosterone axis. Previous data from Yuriko et al. [17] indicated that increased testicular testosterone concentrations after treatment with 8 g of garlic powder was associated with an increase in LH plasma levels. The discrepancies observed in testosterone levels between our present study and the Yuriko's study could be attributed to the different types of garlic preparations used. Indeed, it is possible that crude garlic (present study) and garlic powder [17] do not contain the same active compounds. However, As is most frequently used in its crude form in cooking.

Because a decrease in testosterone levels was observed after crude garlic feeding, it was of interest to examine if the substrate of androgen was affected by the treatment. Indeed, cholesterol is involved in testicular steroidogenesis and is the most important precursor in the synthesis of steroid hormones. In our study, the cholesterol content in testicular tissue remained unchanged. These results suggest that crude garlic might inhibits steroidogenesis by an other way than a decrease in its substrate income. Therefore, one may hypothesize that As inhibits steroidogenesis in three different ways: (i) it might affect free cholesterol mobilization towards Leydig cell mitochondria; (ii) it might disrupt cholesterol mitochondrial translocation, which is an important step of steroidogenesis with the STAR protein as an effector; and (iii) it might prevent cholesterol conversion into testosterone by impairing activities of key regulatory enzymes of steroidogenesis. These hypotheses are currently being investigated.

Testosterone has been shown to be essential for spermatogenesis completion, because it stimulates the conversion of round spermatids into elongated spermatids between stage VII and stage VIII of the spermatogenetic cycle. Androgen deficiency disturbs the spermiation process [18] by altering spermatid-Sertoli cell junctions, which results in premature detachment of round spermatids from Sertoli cells and seminal epithelium [19], along with apoptosis and activation of caspases [20]. In this context, the decrease in plasma and testicular testosterone production observed in the present study might explain the increased percentage of empty seminiferous tubules in As-fed rats. Moreover, decreased testosterone levels have been previously associated with histological alterations in Sertoli and Leydig (androgen target) cells [21]. In this context, the possibility exists that the ultrastructural alterations of Sertoli and Leydig cells observed here were related to the decreased testosterone levels. Therefore, our results are in accordance with the study of Dixit and Joshi [6] who reported a spermatogenesis arrest at the primary spermatocyte stage with 50 mg of garlic powder oral administration for 70 days. We showed here that raw crude garlic feeding impaired male reproductive function and spermatogenesis in male rats. Other data obtained with different garlic preparations has shown that garlic is effective in assisting the recovery of testicular function after experimental testicular hypogonadism [9]. These discrepancies could be related to the type of preparations used (e.g. garlic powder [6, 17], water extract [12], aged garlic, raw garlic juice and heated garlic juice [9]) or the doses and the method of administration (gavage, i.p. injection, ad libitum). The active principle in garlic supporting the inhibitory effect remains to be identified. One molecule, attridium (diallyl trisulfide), is a good candidate because it is known for its spermicidal activity in vitro [7, 8].

In summary, we showed that crude garlic feeding altered the reproductive function in adult male rats in accessory glands (prostate, epididymis and seminal vesicle) and testis (spermatogenesis). This action is probably related to an effect of garlic on the Leydig cells, and perhaps also on the Sertoli cells, with a decrease in serum and testicular testosterone levels and a disruption of normal spermatogenesis.

Acknowledgment

We are grateful to Professor Saad Ali and his staff for technical assistance.

References

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3 Raji Y, Oloyo AK, Morakinyo AO. Effect of methanol extract of Ricinus communis seed on reproduction of male rats. Asian J Androl 2006; 8: 115_21.

4 Al-Majed AA, Al-Yahya AA, Al-Bekairi AM, Al-Shabanah OA, Qureshi S. Reproductive, cytological and biochemical toxicity of Yohimbe in male Swiss albino mice. Asian J Androl 2006; 8: 469_76.

5 Agarwal KC. Therapeutic actions of garlic constituents. Med Res Rev 1996; 16: 111_24.

6 Dixit VP, Joshi S. Effects of chronic administration of garlic (Allium sativum Linn) on testicular function. Indian J Exp Biol 1982; 20: 534_6.

7 Qian YX, Shen PJ, Xu RY, Liu GM, Yang HQ, Lu YS, et al. Spermicidal effect in vitro by the active principle of garlic. Contraception 1986; 34: 295_302.

8 Chakrabarti K, Pal S, Bhattacharyya AK. Sperm immobilization activity of Allium sativum L. and other plant extracts. Asian J Androl 2003; 5: 131_5.

9 Kasuga S, Uda N, Kyo E, Ushijima M, Morihara N, Itakura Y. Pharmacologic activities of aged garlic extract in compari-son with other garlic preparations. J Nutr 2001; 131: 1080S_4S.

10 Allain CC, Poon LS, Chan CS, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem 1974; 20: 470_5.

11 Wang ZP, Gu ZP, Cao L, Xu Y, You GD, Mao BY, Qian SZ. Effects of tripchlorolide on the epididymides and testes of rats. Asian J Androl 1999; 1: 121_5.

12 al-Bekairi AM, Shah AH, Qureshi S. Effect of Allium sativum on epididymal spermatozoa, estradiol-treated mice and general toxicity. J Ethnopharmacol 1990; 29: 117_25.

13 Costello LC, Franklin RB. Testosterone and prolactin regulation of metabolic genes and citrate metabolism of prostate epithelial cells. Horm Metab Res 2002; 34: 417_24.

14 Elzanaty S, Richthoff J, Malm J, Giwercman A. The impact of epididymal and accessory sex gland function on sperm motility. Hum Reprod 2002; 17: 2904_11.

15 Chemes H. The phagocytic function of Sertoli cells: a morphological, biochemical, and endocrinological study of lysosomes and acid phosphatase localization in the rat testis. Endocrinology 1986; 119: 1673_81.

16 Mathur PP, Chattopadhyay S. Involvement of lysosomal enzymes in flutamide-induced stimulation of rat testis. Andrologia 1982; 14:171_6.

17 Oi Y, Imafuku M, Shishido C, Kominato Y, Nishimura S, Iwai K. Garlic supplementation increases testicular testosterone and decreased plasma corticosterone in rats fed a high protein diet. J Nutr 2001; 131: 2150_6.

18 Saito K, O'Donnell L, McLachlan RI, Robertson DM. Spermiation failure is a major contributor to early spermatogenic suppression caused by hormone withdrawal in adult rats. Endocrinology 2000; 141: 2779_85.

19 Beardsley A, O'Donnell L. Characterization of normal spermiation and spermiation failure induced by hormone suppression in adult rats. Biol Reprod 2003; 68: 1299_307.

20 Tesarik J, Martinez F, Rienzi L, Iacobelli M, Ubaldi F, Mendoza C, et al. In-vitro effects of FSH and testosterone withdrawal on caspase activation and DNA fragmentation in different cell types of human seminiferous epithelium. Hum Reprod 2002; 17: 1811_9.

21 Yang ZW , Kong LS, Guo Y, Yin JQ, Mills N. Histological changes of the testis and epididymis in adult rats as a result of Leydig cell destruction after ethane dimethane sulfonate treatment: a morphometric study. Asian J Androl 2006; 8: 289_99.

The effect of a hydro-alcoholic extract of olive fruit on reproductive argons in male sprague-dawley rat.

Abstract

BACKGROUND:

Olive (Olea europea), from the Oleaceae family, is known as a phytoestrogen plant compound, containing Lignans and phenoliccompounds. Some studies have shown phytoestrogens to have spermatogenesis-decreasing effects.

OBJECTIVE:

The present study investigated the effects of a hydro-alcoholic extract of olive fruit on reproductive argons in male rats.

MATERIALS AND METHODS:

The hydro-alcoholic olive (Olea europaea) extract was given orally to three experimental groups of rats in 50, 150, and 450 mg/kg in 48 days. The vehicle group was fed with normal saline and nothing was given to the control group (each group with 8 rats). After 49 days reproductive indicators i.e., sperm count, sperm motility, the weight of prostate, testis, epididymis, and seminal vesicle were measured.

RESULTS:

The results showed a significant decrease in the weights of the left testicle, seminal vesicle, testosterone hormone, sperm count and sperm motility but there was no significant difference with regard to the weights of prostate and epididymis, and estradiol hormone.

CONCLUSION:

This study suggests that olive extract may have deleterious effects on fertility factors; therefore, after further studies, it may be used as a contraceptive in males.

KEYWORDS:

Infertility; Olea Europaea; Phytoestrogens; Spermatogenesis

Introduction

Phytoestrogens plant compounds with biologic-estrogenic activity, structurally similar to 17β-estradiol, are first converted to heterocyclic compounds similar to estrogens in structure and then conjugated in the liver (1-3). Phytoestrogens are categorized into three major classes: Isoflavones, Lignans, and Coumestans (4). These plants are vastly available in food sources like soybean, flax seed, fennel and Actinidia chinensis  (5). Epidemiological studies show that food sources containing phytoestrogens cause lower risk of cardiovascular diseases and also prostate and breast cancers (6).

Australian pastures developed a widespread infertility in the 1940s. A particular type of clover (Trifolium species) , rich in formononetin, is included in the sheep grazing which in the rumen during the process of fermentation will be changed to daidzein (7). Other studies claim that the phytoestrogens present in a type of summer grass reduced the reproduction rate of sparrows and deer in California; these studies also report that young mice fed by their mothers suffered from infertility problems because they were exposed to high amounts of phytoestrogens (8-10). It was also observed that soy bean caused infertility in Cincinnati’s panthers, a problem solved by eliminating soy bean from the food supply (11).

Olive (olea europea), from the oleaseae family, is known as a phytoestrogen plant compound since it contains Lignans and phenolic compounds (12-14). Olive contains stilbenoids, phenolic acidand flavonoids, and, because of the presence of oleuropein, has antioxidant, anti hyperlipidemic and anti-ischemic effects (15). It is also useful in curing gastrointestinal problems since it has laxative effects (16). What’s more, olive is employed in treating dermatological diseases like psoriasis and atopic dermatitis (17).

Additionally, the plant has antimicrobial, antivirus and anti-fungus attributes (18-19). It should be mentioned that olive reduces osteoporosis in Menopausal women (20). Therefore, with regard to the phytoestrogenic effects of this plant, the present study investigated the effects of olive extract on the fertility reduction of male rats.

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Materials and methods

Plant collection and preparation of extract

Olive fruit was collected in summer from Kesht-o-Sanate Bayza Co., (Shiraz, Iran) and the class was specified by an expert to be Olea Europaea L (Voucher Number: 037422) (21). Then the fruit supply was dried in exposure to air and away from sun beam, and after being crushed, was taken to the percolator where it was percolated by means of ethanol 70% (4 times per day, 20cc solvent each time, for 25 days). The resultant ethanol extract was preserved in closed and dark containers in refrigerator until the time of experiment.

Animals treatment

In this experimental study, 40 Sprague-Dowley male rats with the average weight of 200-250 grams and age of 8-10 weeks, divided into 5 groups (Table I). They were kept at the Animal Center of the Shiraz University of Medical Sciences at a temperature of 26±2^oC, a cycle of 12h/12h light/dark. They had access to food and water ad libitum for 49 days. The study adheres to the principles of laboratory care established by Ethics Committee of Shiraz University of Medical Sciences.

Table I

Summary of experimental groups and the diet/drug treatment protocols^a

Before the administration of the first gavage and 24 hours after that of the last one (i.e., in the 49^th day), all the rats were weighed, and blood samples were taken from their tail vein. The blood samples were then centrifuged (1500 rpm, 20 minutes),the serum was separated, and stored at -80^oC for the measurement of estradiol and testosterone, using immunoassay technique. Spectra Testosterone, and estradiol kits were used according to their manufacturer’s instruction (Orion Diagnostica; Finland and DRG Instruments GmbH; Germany).

In the 49^th day and under anesthesia by ether, the rats were dissected and the reproductive organs including the left testicle, epididymis, seminal vesicle and the left prostate were removed and cleaned by physiological serum. After removing lipid remnants, the organs were weighed by a digital scale, and the exact measures were recorded for the following analyses.

Sperm motility

Animals were sacrificed and their reproductive organs were dissected; a length of 1cm of the left end of vas deferens duct was horizontally cut. The location was chosen because of the presence of more mature sperm cells in comparison to the beginning area of the duct (22). The sperm cells were then placed in 5ml of Hanks Balance Salt Solution (HSBB) on the incubator set at 37^oC so that they were evenly distributed.

Then, 250µl of the liquid was taken by a sampler and the motility was measured under a microscope with a magnifying power of 40X as follows: ten spots were randomly chosen; in each, the sperm motility was monitored and measured as one of Grade a (these are the strongest of sperm cells and swim fast in a straight line; sometimes it is also denoted motility 1); Grade b (these also move forward but tend to travel in a curved or crooked motion; sometimes also denoted motility 2); Grade c (they do not move forward despite the fact that they move their tails; sometimes also denoted motility 3); or Grade d (these are static and fail to move at all; sometimes also denoted motility 4) (23).

Sperm count

The sperm samples present in the Hanks medium were loaded on the neubauer hemocytometer for counting the sperm numbers. Then, the sperm count of 1 mm³ of diluted semen was computed by Equation 1:

(1)A=B.C.D

where A and B stand for the total sperm count taken from 1cm of vas deferens, the sperm count of 0.1mm3 of the liquid, respectively, and C and D equal 10 and 5000 mm as the depth and concentration factors, respectively (24).

Statistical analysis

Quantitative data are presented as Mean±SD. Sperm count and motility, of control and experimental groups are compared using one-way analysis of variance (ANOVA), and Tukey test is use to find the statistical differences among their means. P<0.05 is considered to be statistically significant.

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Results

Oral administration of various concentrations of olive extract resulted in no significant difference in the rats’ weights among the control group, the vehicle, and the experimental groups (Figure 1). The weights of the left testicle in the groups administered dosages of 50, 150, and 450 mg/kg and seminal vesicle in the groups administered a dosage of 150 mg/kg showed a significant decrease (p=0.03).

Figure 1

The effect of different dosages of olive extract on rats’ weights. There was no significant difference in the rats’ weights among the control, the vehicle, or the experimental groups

However, there was no noticeable difference with regard to the weights of prostate (p=0.07) and epididymis (p=0.10) (Figure 2).The results of the measuring of the testosterone demonstrate a significant decrease (p≤0.04) in testosterone in the experimental groups in comparison with the control group. The highest decrease was observed in the group administered the 450 mg/kg dosage (Figure 3).

Figure 2

The effect of the olive extract on the weights of prostate, seminal vesicle, left testicle and epididymis. * There was a significant decrease in left testis’ weight in the experimental groups compared to the control and the vehicle groups. ** ...

Figure 3

The effect of olive extract on testosterone levels (ngr/ml). * There was a significant decrease in testosterone levels in the experimental groups compared to the control and the vehicle groups

The results of the measuring of the estradiol, reveal no significant difference among the control, vehicle and/or other experimental groups (p≤0.07) (Figure 4).

Figure 4

The effect of olive extract on estradiol levels (pgr/ml).There was no significant difference in the rats’ weights among the control, the vehicle, or the experimental groups

There was a significant decrease (p≤0.001) in the sperm count of the groups administered dosages of 50, 150 and 450 mg/kg/day in comparison with the control and vehicle groups; the most effective dose was 450 mg/kg/day (Figure 5).

Figure 5

The effect of the olive extract on sperm count. There were significant dosage dependent decreases in the experimental groups compared to the control and the vehicle groups. The decrease enhanced as the dosage level increased. * There was a significant ...

The results of the study of sperm motility show a significant decrease (p≤0.04) in the sperm motility of the groups administered dosages of 50, 150 and 450 mg/kg/day in comparison with the control and vehicle groups (Figure 6).

Figure 6

The effect of olive extract on sperm motility in different groups

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Discussion

Phytoestrogens are plant compounds with structures and functions similar to those of 17-β estradiol, which produce effects like those by estrogen (3). The olive, as it contains phenol compounds, is one of the natural plants rich in phytoestrogens, and belongs among the Lignans (13-14). The plant can highly decrease menopausal syndrome in women (25). It also decreases the occurrence of colorectal, prostate and breast cancers (6). The findings of the present study show that olive decreases the levels of reproductive indicators such as sperm count and motility, testosterone, the weights of testicle and seminal vesicle in male rats. The results of the study showed no change in the rats’ weights; therefore, it can be concluded that the extract produces no effect on metabolism.

The results also show a significant decrease in testosterone level among the five groups, which is dependent on the concentration of the extract; the decrease in testosterone is positively correlated to the concentration of the extract. Studies by Webber et al and Roberts et al on the effects of phytoestrogens on testosterone support these results. McGravy et al found that the LH level in rats decreases as a result of exposure to Genistein. According to their study, it is possible that Phytoestrogen has an inhibitory effect on the enzyme 17 β-hydroxy steroid hydrogenase human type 5; therefore, the synthesis of testosterone in adrenal cortex reduced (27-29)

The results of the study show no significant differences of estradiol levels among the groups. Although, studies by Webber etal and Glazier and Boman have also shown that phytoestrogens produce no significant decrease in estradiol levels, and a study in 2005 about the effect of Actinidia Chinensis on male rats’ spermatogenesis showed an increase in estradiol (27, 30, 31). It should also be noted that Actinidia Chinensis belongs to Genisteins while olive is from Lignan group, which can justify the discrepancy of the results of different experiments as a result of the different types of phytoestrogen under study and the differences in the concentrations employed.

The administration of the olive extract in all the three concentrations resulted in a significant decrease in both sperm count and sperm motility. Roberts et al also reported similar results (29). One study reported that Genestein phytoestrogen inhibit tyrosine kinase enzymes, which accordingly results in the decrease of sperm count and sperm motility (32). Another study on fennel and Actinidia Chinensis showed similar results (31). On the other hand, there are some studies which have claimed that a phytoestrogen-based regiment has no effect on the quality of mature sperm cells (33, 34). All in all, it can be said that the way a phytoestrogen affects sperm quality depends on its type.

Our findings show significant decreases of the weights of the left testicle and seminal vesicle in three of the administered dosages, but no significant difference in the weights of the left epididymis and prostate. A study by Sprando et al showed a decrease in the weights of testicle and seminal vesicle. It should be noted that flax seed is from the Genisteins family (35). Different studies; however, have reported the effects of phytoestrogens on the weights of reproductive organs differently (36).

With regard to the explained results, there is this possibility that the different effects of phytoestrogens on the male productive system is due to estrogenic and anti-estrogenic effects, as phytoestrogens function through estrogen receptors which have both agonistic and antagonistic properties. Depending on the type of phytoestrogen and the location, the effects can differ. For example, Isoflavones are very weak agonists which bind to estrogen receptors less than estradiol does (37).

When estradiol levels are low in the body and binding is therefore less competitive, Isoflavones show stronger agonistic effects. On the other hand, the anti-estrogenic effects of Isoflavones are co-dependent on relative concentrations of endogenous phytoestrogens and estrogens, and it is quite possible that when estrogen is high, phytoestrogens make estradiol receptors unavailable to estradiol. Genisteins can also have both estrogen-like and anti-estrogen-like properties (because of the competitiveness in binding to proteins) (37).

Phytoestrogens produce various physiological effects in both the human body and animal models. Their effects on the male reproductive system depend on the type of the phytoestrogen, concentration and the model under study (17).

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Conclusion

In conclusion, olive fruit extract significantly decreased fertility parameters in the male adult rat. However, it is needed more study about the mechanism by which olive fruit extract creates its anti-fertility effects on human being which are still unknown. Nevertheless, considering our findings in this animal model, it is recommended that the olive fruit extract maybe used in the future as a contraceptive in males.

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Acknowledgments

The authors wish to thank the Shiraz University of Medical Sciences, and also Mr. Izad Noori for her excellent technical supports.

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Conflict of interest

None of the authors have any potential conflict of interest of a funding source for this study.

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