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Protective Role of Royal Jelly in Oxymetholone-induced Oxidative Injury in Mouse Testis

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ABSTRACT Background: An adverse effect of oxymetholone (OXM), an anabolic-androgenic steroid used as energetic medicine, is reproductive toxicity. Royal jelly (RJ) is an efficient antioxidant that has been used to treat reproductive problems. In this study, we investigated the effects of RJ on OXM-induced oxidative injuries in mouse testes. Methods: Male mice were divided into four groups. Two groups of mice were administered OXM (5 mg/kg/day, p.o.) for 28 days. One of these groups received RJ (100 mg/kg/day, p.o.) concurrently. A vehicle-treated control group and a RJ control group were also included. Results: The OXM-treated group showed a significant decrease in the serum testosterone concentration and spermatogenic activities, along with many histological alterations. OXM treatment also caused a significant decrease in catalase activity with an increase in lipid peroxidation in the mouse testes. The above-noted parameters were restored to near normal levels by RJ co-administration. Conclusion: The results demonstrate that RJ protects against OXM-induced reproductive toxicities. Keywords: Mouse, Oxymetholone, Royal Jelly, Testis.
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Iranian Journal of Toxicology Volume 8, No 25, Summer 2014
1. Department of Basic Sciences, Urmia University, Urmia, Iran.
2. Department of Biology, Urmia University, Urmia, Iran.
*Corresponding Author: E-mail:
Protective Role of Royal Jelly in Oxymetholone-induced Oxidative
Injury in Mouse Testis
Gholamreza Najafi1, Vahid Nejati2, Ali Shalizar Jalali*1, Ensieh Zahmatkesh2
Received: 01.10.2013 Accepted: 30.11.2013
Background: An adverse effect of oxymetholone (OXM), an anabolic-androgenic steroid
used as energetic medicine, is reproductive toxicity. Royal jelly (RJ) is an efficient
antioxidant that has been used to treat reproductive problems. In this study, we
investigated the effects of RJ on OXM-induced oxidative injuries in mouse testes.
Methods: Male mice were divided into four groups. Two groups of mice were
administered OXM (5 mg/kg/day, p.o.) for 28 days. One of these groups received RJ (100
mg/kg/day, p.o.) concurrently. A vehicle-treated control group and a RJ control group were
also included.
Results: The OXM-treated group showed a significant decrease in the serum
testosterone concentration and spermatogenic activities, along with many histological
alterations. OXM treatment also caused a significant decrease in catalase activity with an
increase in lipid peroxidation in the mouse testes. The above-noted parameters were
restored to near normal levels by RJ co-administration.
Conclusion: The results demonstrate that RJ protects against OXM-induced
reproductive toxicities.
Keywords: Mouse, Oxymetholone, Royal Jelly, Testis.
IJT 2014; 1073-1080
Worldwide, up to 20% of couples are
infertile. Approximately 30-50% of human
infertility is attributable to male infertility [1,
2]. Although hormonal causes of male
infertility are well known, lack of sufficient
knowledge about intracellular mechanisms
leading to the production of important factors
necessary for regulating spermatogenesis is
the main reason behind the inability to
diagnose and treat certain forms of idiopathic
infertility [3]. Testis, the main organ of the
male reproductive system, consists of two
parts: the testis parenchyma and intercellular
substances. The parenchyma is composed of
long and coiled tubes called seminiferous
tubules. As the seminiferous tubules comprise
approximately 80% of the testicular mass, the
morphological measurements of seminiferous
tubules are important in the studies of testis
tissue [4-7].
Anabolic-androgenic steroids (AASs)
are used by a considerable proportion of the
community to enhance their physique and
performance. More than one million of
Americans use or have used AASs [8].
Oxymetholone (OXM) is an active nutritional
17α-alkylated anabolic-androgenic steroid
derived from testosterone [9]. Since OXM
can stimulate erythropoiesis and increases
erythropoietin production, it is used for the
treatment of anemias caused by low red cell
production [10]. In addition, this drug is
currently applied to the treatment of
myelotoxic anticancer drugs-induced
myelofibrosis [9] and AIDS-associated
wasting [11]. Despite its therapeutic
significance, it has been reported that OXM
causes hepatic and cardiac damages [12, 13]
as well as reproductive toxicities in humans
and experimental animals [14-16]. Also,
AASs have been found to induce
hypogonadotrophic hypogonadism via
negative feedback to the hypothalamus
leading to testicular atrophy and impaired
spermatogenesis [17, 18].
Iranian Journal of Toxicology Gholamreza Najafi et al
1074 Volume 8, No 25, Summer 2014;
Royal jelly (RJ), a secretion product of
the hypopharyngeal and mandibular glands of
nurse bees, is a mixture that contains many
important compounds with biological activity
such as free amino acids, proteins, sugars,
fatty acids, minerals, and vitamins [19]. Due
to its complex composition, RJ possesses
numerous pharmacological properties
including antioxidant, anti-inflammatory,
antitumor, anti-allergic, antibiotic,
hypotensive, neurotrophic, and
immunomodulatory activities [20-23].
Furthermore, independent studies have
indicated that RJ has positive effects on the
reproductive system and fertility in humans
and animals [24-27]. Hence, the present study
was designed to investigate the probable
protective effects of RJ on OXM-induced
oxidative injuries in mouse testes.
This study was carried out on healthy
adult sexually mature male (9 weeks of age)
mice weighing 30±2 g. A total of 32 mice
were obtained from the Animal House of
Faculty of Science, Urmia University, Urmia,
Iran. The animals were housed in filter-top
polycarbonate cages in an air-conditioned
room (temperature: 25±2 ◦C, relative
humidity: 50±10%, and 12 h light/12 h dark
photoperiod) free from any sources of
chemical contamination with free access to
standard diet and water throughout the
experimental period. The experimental
protocol and procedures used in this study
were approved by the Ethics committee of the
Urmia University, Urmia, Iran for the care
and use of laboratory animals.
Experimental design
After seven days of acclimation to the
environment, the mice were randomly divided
into four treatment groups of eight animals
each (n = 8) and treated orally for 28 days as
Group I (Control): received saline vehicle
(0.1 ml/mice)
Group II (OXM): OXM (5 mg/kg)
Group III (RJ): royal jelly (100 mg/kg)
Group IV (OXM + RJ): royal jelly (100
mg/kg) + OXM (5 mg/kg).
Animals were euthanized by CO2
exposure in a special device following
anesthesia with ketamine (75 mg/ kg, IP) 24
hours after the last treatment. Blood was
collected from atrium in routine biochemical
test tubes without anticoagulant for
serological analysis. The abdominal cavity
was opened up through a midline abdominal
incision and the testes were excised quickly.
One half of the right and left testes were fixed
in Bouin’s fixative (0.2% picric acid/2%
(V/V) formaldehyde in PBS) for histological
evaluation and the other halves were kept
frozen at -70 °C until they were homogenized
for further biochemical studies.
Assessment of catalase activity
Catalase (CAT) activity in
homogenized testicular tissue was determined
according to Aebi (1984) [28]. Its activity was
assayed by determining the rate of
degradation of hydrogen peroxide at 240 nm
in 10 mM of potassium phosphate buffer (pH
7.0). Extinction coefficient of 43.6 mM/cm
was used for calculation. One unit is defined
as 1 pmol of hydrogen peroxide consumed
per minute, and the specific activity is
reported as units/mg of protein.
Assessment of lipid peroxidation
Lipid peroxidation (LPO) was
determined by the spectrophotometric TBA
assay as previously described [29].
Malondialdehyde (MDA), formed as an end
product of the peroxidation of lipids, reacts
with thiobarbituric acid (TBA) to generate a
colored product that can be measured
optically at 532 nm. Results were expressed
as µmol MDA/mg protein.
Testosterone assessment
Serum concentration of testosterone
was measured by enzyme-linked
immunosorbent assay (ELISA) as described
in the instructions provided by manufacturer’s
kit (Demeditec Diagnostics GmbH,
Histological analysis
After fixation of testes, they were
dehydrated through a gradual series of alcohol
Protective Role of Royal Jelly in Oxymetholone… Iranian Journal of Toxicology
1075; Volume 8, No 25, Summer 2014
and cleared in three changes of xylene before
embedded in paraffin. Thin sections (5µm)
perpendicular to the longest axis of the testis
were cut using a microtome and stained with
hematoxylin and eosin according to the
standard method. Histological analysis was
performed under light microscope in terms of
the changes in different groups as compared
to the control group.
Determination of histological parameters
For each testis, five vertical sections
from the polar and the equatorial regions were
sampled [30] and an unbiased numerical
estimation of the following histological
parameters was determined using a systematic
random scheme.
Seminiferous tubules diameter (STsD)
and interstitial tissue thickness (ITT): For
measuring STsD and ITT, 200 round or
nearly round cross-sections of seminiferous
tubules were randomly analyzed in each
mouse (one hundred per testis). Then, two
perpendicular diameters of each cross-section
of seminiferous tubules were measured using
an ocular micrometer of light microscopy
(Olympus Co., Germany) and their means
were calculated. Also, ITT was measured in 4
equidistance of each cross-section of
seminiferous tubules and their means were
calculated [31].
Number of mononuclear immune cells
(MNICs) in testicular tissue: The number of
MNICs was determined by using the unbiased
counting frame proposed by Gundersen
(1977) [32].
Tubule differentiation index (TDI) and
spermiation index (SPI): 200 cross-sections of
seminiferous tubules were randomly analyzed
in each mouse (one hundred per testis) for the
calculation of TDI and SPI. TDI is the
percentage of seminiferous tubules containing
at least three differentiated germ cells [33].
SPI is the percentage of seminiferous tubules
with normal spermiation [34].
Sertoli cell index (SCI) and mitotic
index (MI): Sixty seminiferous tubules per
group were randomly examined for the
calculation of SCI and MI. SCI is the ratio of
the number of germ cells to the number of
Sertoli cells identified by a characteristic
nucleus and nucleolus in all seminiferous
tubules [35]. MI, the number of round
spermatids for each pachytene primary
spermatocytes, was calculated for
determination of cell loss percentage during
cell division [36].
Statistical analysis
The results are expressed as the mean ±
standard error of mean (S.E.M.). Differences
between the groups were assessed by the
analysis of variance (ANOVA) using SPSS
software package for Windows. Statistical
significance between groups was determined
by Tukey’s multiple comparison post hoc test
and the P-values less than 0.05 were
considered to be statistically significant.
Antioxidant status study
The effects of different treatments on
CAT activity and MDA level in testis are
depicted in Table 1. Treatment with OXM
alone resulted in a significant decrease in
CAT activity in testis tissue, whereas it
caused a significant increase in MDA in the
same tissue. Treatment with RJ in
combination with OXM resulted in a
significant improvement in these parameters
in testis tissues compared to the OXM alone
group (Table 1).
Table 1. Effect of oxymetholone and Royal jelly on the testis antioxidant status.
CAT (U/mg pt.)
51.6±0.6 23.1±0.2
MDA (µmol/mg pt.) 3.9±0.1 6.2±0.2
The values are expressed as mean ± S.E.M. (n = 8).
a Significant differences as compared with the control group at P <0.05
b Significant differences as compared with the OXM group at P <0.05
Iranian Journal of Toxicology Gholamreza Najafi et al
1076 Volume 8, No 25, Summer 2014;
Testosterone level
Results of testosterone concentrations
study (Figure 1) revealed that treatment with
OXM alone caused a significant decrease in
serum testosterone level as compared to the
control group. The administration of RJ along
with OXM significantly restored serum
testosterone level towards the control value
(Figure 1).
Figure 1. Effect of oxymetholone and Royal jelly on serum concentrations of testosterone.
The values are expressed as mean ± S.E.M. (n =8).
a Significant differences as compared with the control group at P <0.05
b Significant differences as compared with the oxymetholone group at P <0.05
Histological parameters
As seen in Table 2, treatment of male
mice with OXM caused a significant decrease
in seminiferous tubules diameter (STsD),
while interstitial tissue thickness (ITT)
increased compared to that of control. Co-
administration of RJ significantly attenuated
the OXM-induced morphometric changes.
Data exist in Figure 2 revealed that
infiltration of mononuclear immune cells
(MNICs) in testicular tissue was significantly
elevated by OXM treatment. However, this
elevation in the number of MNICs was
inhibited by concurrent treatment with RJ.
Moreover, OXM treatment induced the
deletion of germ cells during
spermatogenesis, which resulted in significant
decreases in TDI and SCI (Table 3). Due to
the germ cells deletion, SPI and MI were also
greatly decreased in the OXM-treated mice
(Table 3). RJ treatment significantly
prevented the OXM-induced germ cell loss
from seminiferous tubules (Table 3).
Histopathologic findings
There were no marked histological
alterations in testes of control (Figure 3a) and
RJ-only (Figure 3b) groups, while drastic
morphologic changes were observed in the
testis of OXM-treated mice (Figure 3c). The
seminiferous tubules of these animals showed
severe hypocellularity and intraepithelial
vacuolization and were displaced by some
fibrinoid debris. Rupture, vacuolization,
inflammatory cells infiltration, and interstitial
space widening were also observed in
intertubular connective tissue of testes
following OXM treatment (Figure 3c). OXM-
induced lesions in testicular tissue were
greatly recovered by RJ co-administration,
although partial disorganizations were
observed in some seminiferous tubules
epithelium (Figure 3d).
Table 2. Effect of oxymetholone and Royal jelly on histological parameters of testis.
STsD (µm) 204.75±6.55 149.36±6.07
ITT (µm)
48.50±5.02 155.83±9.27
STsD, seminiferous tubules diameter; ITT, interstitial tissue thickness.
The values are expressed as mean ± S.E.M. (n = 8).
a Significant differences as compared with the control group at P <0.05
b Significant differences as compared with the OXM group at P <0.05
Control OXM Royal Jelly OXM+Royal Jelly
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Table 3. Effect of oxymetholone and Royal jelly on spermatogenic activities.
TDI (%) 65.83±4.40 41.66±2.20
SPI (%) 70.88±2.73 41.83±1.73
SCI (%) 63.73±0.66 47.16±3.10
MI (%) 68.61±8.28 38.88±1.46
TDI, tubule differentiation index; SPI, spermiation index; SCI, Sertoli cell index; MI, miotic index.
The values are expressed as mean ± S.E.M. (n = 8).
a Significant differences as compared with the control group at P <0.05
b Significant differences as compared with the OXM group at P <0.05
Figure 2. Effect of oxymetholone and Royal jelly on numbers of mononuclear immune cells
(MNICs) in testicular tissue.
The values are expressed as mean ± S.E.M. (n = 8).
a Significant differences as compared with the control group at P <0.05
b Significant differences as compared with the oxymetholone group at P <0.05
Figure 3. Cross-sections of testes in mice treated with oxymetholone and/or royal jelly.
Testes from control (a) and RJ-treated (b) mice show intact seminiferous tubules with active
spermatogenesis. However, a testis from an OXM-treated mouse (c) reveals germ cells deletion and impaired
spermatogenesis. In the testis of a mouse treated with OXM and RJ, the seminiferous epithelium is less
abnormal as compared with (c), although partial disorganizations appear in some seminiferous tubules
Hematoxylin and eosin (×600)
Control OXM Royal Jelly OXM+Royal Jelly
Number of MNICs/mm2
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1078 Volume 8, No 25, Summer 2014;
Synthetic AASs do the same work as
androgenic hormones do. Evidence exists that
these compounds have profound effects on
male endocrinological and reproductive
systems [37]. It has been reported that AASs
induce oligozoospermia and azoospermia (18)
as well as sperm morphology impairment
[16]. Previous study on male rats has also
confirmed the potential of AASs for causing
spermatogenic arrest [14]. Although the
mechanism by which AASs cause
reproductive toxicities is still under debate,
impaired spermatogenesis is supposed to
occur because of AASs-induced
hypogonadotrophic hypogonadism [17, 18].
Spermatogenesis is a complex process,
where spermatogonia develop into highly
differentiated spermatozoa through several
strictly controlled steps [38] and histological
parameters, such as STsD and ITT along with
TDI, SPI, SCI and MI, can give information
about the testicular damage degree as a
consequence of germ cell death. In general,
massive germ cell loss caused by reproductive
toxicity is followed by considerable
alterations in testicular histological
parameters. As shown in the present study,
depletion of seminiferous epithelium and the
consequent changes in histological
measurements caused by OXM were
confirmed in our report.
In the present study, OXM treatment
caused a marked reduction in serum
testosterone level, confirming a previous
report that AASs, especially 17α-alkylated
steroids, induced marked depressions of
serum testosterone and sex hormone-binding
globulin by means of androgenic receptors
occupation [39].
Sertoli cells (SCs) are the main somatic
cells of testis which play a major role in
cytoarchitectural organization of the
seminiferous tubules and, more importantly,
govern the differentiation of germ cells
(GCs). The physical and functional supports
of SCs are essential for GCs survival and
development [1, 2]. Therefore, a potential
explanation for the failure of spermiogenesis
in the OXM-treated mice is disruption of
testosterone-dependent junction of SCs with
GCs leading to their disorganization and
MNICs infiltration in testicular tissue is
an indicator of inflammation, a complex
clinical condition that can negatively affect
reproductive potential, which shows some
alterations occur at the suspected testis.
Hence, the elevation in MNICs infiltration
observed in testicular tissue of OXM-treated
animals might reflect the role of OXM in
induction of inflammation.
To date, several reports have supported
the fact that RJ has a predominant repro-
protective role in humans and animals.
Recently, it has been revealed that oral
administration of RJ counters “summer
infertility” in male rabbits [24]. Furthermore,
results of a study on 99 couples with
asthenospermia-induced infertility have
revealed that a simple and efficient way of
treating this condition is the intravaginal
administration of RJ and honey [25].
Additionally, it has been found that RJ has a
potential positive effect on development of
genital organ in male mice due to its highly
efficient anti-oxidant properties [27].
In the present study, RJ co-
administration provided effective protection
against OXM-induced oxidative injuries in
mouse testis. This reinforces the fact that RJ
as a potent and safe antioxidant has beneficial
effects against oxidative stress–related
toxicities. The reason why RJ co-
administration attenuated OXM-induced
reproductive toxicity could be attributed to
the fact that it contains spermatogenesis-
stimulating substances such as vitamin C,
vitamin E, and arginine [40]. Moreover, it has
been demonstrated that RJ inhibits the
production of pro-inflammatory cytokines by
activated macrophages [41].
In sum, it can be concluded that RJ has
a protective effect against OXM-induced
reproductive toxicities through restoration of
antioxidant defense system.
The authors gratefully acknowledge the
financial assistance of Urmia University in
performing this investigation.
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... However, in the group receiving Roy, a protective effect on seminiferous tubules against oxidative stress toxicity caused by oxymetholone was detected. It also increased serum levels of Tes following a restorative effect on interstitial tissue.16 Al-Lahham and coworkers assessed the oxidative effects of Nic and the role of Nicotiana tabacum L. with antioxidative features. ...
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Background: Nicotine administration can generate severe oxidative stress and lipid peroxidation. Royal jelly, with its antioxidant properties, acts as a scavenger of reactive oxygen species. This study describes the apitherapy effects of royal jelly on testicular damage following nicotine administration. Methods: Forty-eight male BALB/c mice were divided into 8 groups (n = 6): saline, 3 different doses of royal jelly (100, 150, and 200 mg/kg body weight (BW) per day), nicotine (1.5 mg/kg), and 3 different groups of Nic + Roy (1.5 mg/kg of Nic + 100, 150, and 200 mg/kg BW per day of royal jelly). Nicotine was administrated intraperitoneally, and royal jelly was prescribed orally for 10 consecutive days. Serum levels of hormones (testosterone, luteinizing hormone, and follicle-stimulating hormone), total antioxidant capacity, nitric oxide (NO) status, malondialdehyde levels, sperm DNA fragmentation, sperm parameters, histopathological changes (H&E staining), immunohistochemistry against apoptotic proteins, and gene expression of Bcl-2, p53, Caspase-3, and Nrf2 (real-time PCR) were assessed to evaluate the molecular and histological changes. Results: Hormone levels, sperm parameters, and status of antioxidants were decreased significantly (p < .05) following nicotine administration. Moreover, royal jelly treatment normalized hormonal and antioxidant characteristics, decreased apoptotic gene expression, increased Nfr2 gene expression, and restored histopathological alteration to the physiological status significantly (p < .05). Conclusion: Royal jelly upregulates the antioxidant status, inhibits the mitochondrial-dependent apoptosis pathway, and increases the rate of proliferation. This therapeutic agent effectively protected the testis against nicotine-associated damages by antioxidant and anti-apoptotic effects.
... The approach to pollinator maintenance differs between European countries, and the focus of the conservation strategy [36]. It has the potential to treat human diseases due to its antioxidant, antiaging, antitumor, and anti-inflammatory activity [37][38][39]. ...
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Over the past two decades, there has been an alarming decline in the number of honey bee colonies. This phenomenon is called Colony Collapse Disorder (CCD). Bee products play a significant role in human life and have a huge impact on agriculture, therefore bees are an economically important species. Honey has found its healing application in various sectors of human life, as well as other bee products such as royal jelly, propolis, and bee pollen. There are many putative factors of CCD, such as air pollution, GMO, viruses, or predators (such as wasps and hornets). It is, however , believed that pesticides and microorganisms play a huge role in the mass extinction of bee colonies. Insecticides are chemicals that are dangerous to both humans and the environment. They can cause enormous damage to bees' nervous system and permanently weaken their immune system , making them vulnerable to other factors. Some of the insecticides that negatively affect bees are, for example, neonicotinoids, coumaphos, and chlorpyrifos. Microorganisms can cause various diseases in bees, weakening the health of the colony and often resulting in its extinction. Infection with microorganisms may result in the need to dispose of the entire hive to prevent the spread of pathogens to other hives. Many aspects of the impact of pesticides and microorganisms on bees are still unclear. The need to deepen knowledge in this matter is crucial, bearing in mind how important these animals are for human life.
... Insect as biological resources Najafi et al. 2014, 2. Imai et al. 2012, 3. Taavoni et al. 2014, 4. Seyyedi et al. 2016, 5. El-Nekeety et al. 2007, 6. Silici et al. 2009, 7. Kanbur et al. 20098. Hashimoto et al. 2005, 9. Hattori et al. 2006, 10. ...
The semi‐organized insect industry of India primarily includes apiculture, sericulture and lac culture. In the last two decades the production of raw silk, including mulberry and “vanya silk”, has doubled. An increasing trend for the country has also been observed with regard to honey and beeswax production. India, moreover, stands in the frontline for lac production, but there is immense scope to expand insect‐based ventures. For example, sericultural products other than silk come to mind and with regard to apiculture honey, and beeswax need not be the only bee‐based commercial items. Insects other than honey bees can be used as food and feed or in the decomposition of waste; some species can be reared and even domesticated as pollinators. In this paper we discuss these unexplored sections, which could open up promising possibilities for the insect‐based industry in India as well as other countries.
Oxymetholone is one of the androgenic anabolic steroid medications used primarily to treat anemia. The other clinical applications of oxymetholone include treatment of osteoporosis as well as stimulating muscle growth in malnourished or underdeveloped patients. Oxymetholone was first described and introduced for medical use in 1959 but shortly after was discontinued due its high lipid toxicity in the year 1961. It has strong anabolic effects and weak androgenic effects. Side effects of oxymetholone include increased sexual desire as well as symptoms of masculinization like acne, increased hair growth, and voice changes. Oxymetholone has been said to be one of the most powerful androgenic anabolic steroids available for medical use. It has been proposed that prolonged use of oxymetholone may cause liver-cell damage or hyperplasia, carcinogenicity and reproductive toxicity.
Royal jelly is essential for the fertility of honey bees, but it has a fertility enhancer effect in mammals, too. Rodent experiments proved the mechanisms of action of royal jelly on fertility, including antioxidant, sex hormone, and antiinflammatory effects. In farm animals (chicken, rabbit, buffalo, and sheep), the application of royal jelly contributes to improving the rates of pregnancy and birth and enhances the success rate of in vitro fertilization methods. Human studies proved that royal jelly has a beneficial effect on sperm count and motility and improves the fertilizing ability of male gametes. However, no reliable clinical data support the fertility-improving effect of royal jelly in women, although used widely during assisted reproductive processes. Beyond royal jelly, drone milk has a promising effect on fertility in both sexes in rats, but the clinical evidence is still missing. Further clinical studies are required to explore the real potency of these apiary products in the enhancement of human fertility.
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Background: Sertraline is one of the most widely used antidepressants in the world. The present study was performed to evaluate the protective effects of vitamin E against sertraline-induced damage on tes-ticular tissue in mice. Materials and Methods: In this experimental study, 40 adult male mice were divided into eight groups of 5. The first control group, received 0.3 ml of normal saline per day by gavage. The second, third and fourth groups received sertraline at 5, 10 and 20 mg/kg body weight per day by gavage, respectively. The fifth group received vitamin E at 100 international units per kg body weight per day by gavage. The sixth, seventh, and eighth groups received sertraline at 5, 10, and 20 mg/kg body weight, along with vitamin E at 100 international units per kg body weight per day by gavage, respectively. After 42 days of treatment, testicular specimens were collected and used for histomorphological, histomorphometrical and histochem-ical studies. The obtained data were analyzed in SPSS v. 19 using one-way Analysis of Variance (ANO-VA) and Tukey’s test at the significance level of (p<0.05). Results: Sertraline caused destructive histomorphological and histochemical changes in testicular tissue and also significantly decreased the diameter of the seminiferous tubules, the height of the germinal epi-thelium, the number of spermatocytes, the number of Leydig and Sertoli cells and the Johnsen’s score compared to the control group (p<0.05). Vitamin E administration improved the histomorphological, his-tochemical condition and significantly increased the histomorphometric parameters in testicular tissue (p<0.05). Conclusion: Vitamin E can improve the adverse effects on histomorphological, histomorphometrical and histochemical parameters in the testicular tissue of mice receiving sertraline.
Sperm fertility decreases significantly after freezing. Providing a suitable and useful diluent compound for freezing ram sperm can increase the efficiency of artificial insemination and consequently, the reproductive performance of sheep. Various biological properties such as antibacterial, anti-cancer, immunosuppressive, antioxidant and reproductive properties of royal jelly (RJ) are well known. The aim of this study was to investigate the possible synergistic effect of royal jelly in combination with glycerol and dimethyl sulfoxide (DMSO) in sperm cryopreservation extender of Romanov ram. The pooled semen samples from 5 Romanov rams were allocated into 3 experiments. The effect of 6% DMSO, 6% glycerol and a combination of 3% DMSO + 3% glycerol co-supplemented with 1, 2 and 3% RJ was evaluated in 3 experiments. Samples were frozen by conventional slow freezing method and post-thaw parameters of total motility, progressive motility, plasma membrane integrity, DNA damage, apoptosis, enzyme activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx), total antioxidant capacity (TAC) and malondialdehyde (MDA) were evaluated. The results showed that the percentage of motility, progressive motility, TAC, GPx, SOD and all sperm kinematic parameters except LIN in the group containing 2% RJ + 6% DMSO was higher than the control group (p<0.05). Some parameters such as progressive motility, sperm membrane integrity, TAC, GPx, VAP, VCL, STR and SRT in the group containing 2% RJ + 6% DMSO were more than (p <0.05) in the sperm group containing 1% RJ + 6% DMSO. MDA values in sperm groups containing 2% RJ + 6% DMSO were significantly (p<0.05) lower than the sperm containing 1% royal jelly and the control group. In the sperm group containing 2% RJ + 6% glycerol, sperm membrane integrity, TAC, GPx, SOD, progressive motility and all sperm kinematic parameters except VAP were higher and MDA values and sperm abnormalities were lower than the control group (p<0.05). The sperm group containing 1% RJ and 3% DMSO + 3% glycerol had higher motility, progressive motility, membrane integrity, and all sperm kinematic parameters except VSL; and lower sperm abnormalities, DNA damage, apoptosis and MDA than the control group (p<0.05). As a general conclusion of this study, the addition of 2% RJ + 3% DMSO and 3% glycerol to the freezing extender improved microscopic and biochemical ram sperm parameters after the freeze-thaw process. Hence, moderate concentrations of royal jelly (2%) are sufficient to protect sperm from freezing damage, and high (3%) and low (1%) concentrations do not have a good cryoprotective effect.
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In this study, the protective role of royal jelly (RJ) against the potential toxic effects of sodium benzoate was investigated in Allium cepa L. test material with physiological, genetic, and biochemical parameters. Physiological changes were evaluated by determining weight gain, rooting percentage, root length, and relative injury rate. The genetic evaluations were carried out with chromosomal abnormalities (CAs), micronucleus (MN), tail DNA formation, and mitotic index (MI) ratio parameters. The biochemical evaluations were carried out by determining lipid peroxidation and antioxidant enzyme activities by determining levels of malondialdehyde (MDA), glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT). Further, the interaction of sodium benzoate with antioxidant enzymes was evaluated with molecular docking analysis. The antimutagenic effect of RJ was evaluated as the inhibition of chromosomal abnormalities (CAs) and tail DNA formations. A total of six groups were formed in the study. A. cepa L. bulbs in the control group were treated with tap water; the bulbs in the administration groups were treated with sodium benzoate (100 mg/L), RJ (25 mg/L and 50 mg/L doses), and sodium benzoate–RJ combinations with these doses for 72 h. As a result, it was determined that sodium benzoate application caused inhibition of physiological parameters and MI; induced MN, CAs, and DNA damage; and also caused oxidative stress. Depending on the concentration of RJ application, it reduced sodium benzoate toxicity by showing therapeutic effects in all these parameters. Also, the interaction of sodium benzoate with antioxidant enzyme residues was determined by molecular docking analysis. As a result, it has been understood that abandoning the use of sodium benzoate will be beneficial for the environment and human health and concluded that the use of RJ in the daily diet will be effective in reducing the impact of exposed toxic ingredients.
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Background & objectives: The present study investigated the effects of royal jelly on lead acetate induced toxicity on sperm parameters, reproductive hormone assay, and bak gene expression in NMRI male mice. Methods: In this study, fifty four male mice were randomly divided into nine groups: control group (without royal jelly) (n=6); sham group(10 ml normal saline) (n=6); lead group (1000 ppm, oral) (n=6); Group 4: royal jelly (100 mg/kg/day, oral) (n=6); Group 5: royal jelly (250 mg/kg/day, oral)(n=6); Group 6: royal jelly (500 mg/kg/day, oral)(n=6); Group 7: royal jelly (100 mg/kg/day, oral) + 1000 ppm lead (n=6); Group 8: royal jelly (250 mg/kg/day, oral) + 1000 ppm lead (n=6) and Group 9: royal jelly (500 mg/kg/day, oral) + 1000 ppm lead (n=6). On day 35, blood samples were collected from anaesthetized mice by cardiac puncture to assess reproductive hormones and the testes were harvested for determination of sperm parameters and expression bak gene. Sperm parameters including motility, viability, DNA damage, morphology and total antioxidant capacity (TAC) levels were determined. Results: The results showed that administration of royal jelly significantly enhanced sperm parameters and all reproductive hormone levels compared to control mice, (p<0.05). Also, treatment with lead acetate caused a significant reduction in levels of all reproductive hormones and a significant diminution in sperm motility, morphology, viability; with an increase in percentage of dead spermatocytes (p<0.05). The co-administration of the 250 and 500 mg/kg/day royal jelly with lead acetate could ameliorate the deleterious effects of lead acetate resulting in a significant increase in sperm parameters and all reproductive hormones and increase the total antioxidant capacity (TAC) levels (p<0.05). Also, the expression of bak gene in all treated (sham, royal jelly groups) and control groups was significantly lower than the lead acetate group (p<0.05). Conclusion: In conclusion, our findings suggest that the royal jelly has a beneficial effect on male reproductive parameters following lead acetate induced toxicity in mice.
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Testicular heat stress (HS) can lead to testicular tissue destruction and spermatogenesis disturbances. Royal Jelly (RJ) has been introduced as a potent antioxidant. We investigated the effects of RJ on testicular tissue, oxidative stress and sperm apoptosis in HS-exposed rats. Compared to HS-exposed groups, RJ co-treatment could improve testosterone reduction and histopathological damages. The RJ co-administration decreased MDA level in testicular tissue, while TAC and CAT levels were remarkably increased compared to HS-exposed groups. Moreover, significant higher expression level of Bcl-2 and lower expression levels of P53 and Caspase-3 were seen following RJ co-administration compared to HS-exposed groups. Our data suggest that RJ can effectively ameliorate experimental HS-induced testiculopathies in rats through testicular antioxidant defense system restoration and germ cells apoptosis regulation.
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The study was designed to investigate the efficacy of treating the adult male rats with royal jelly (1g/kg B. Wt. orally) for one month with or without hydrogen peroxide (0.5%) in drinking water on sexual efficiency, glutathione and malondialdehyde tissue testis levels. The current study demonstrated that male rats receiving hydrogen peroxide caused a significant decrease (P<0.05) in the sperm count, percentage of live sperm and glutathione level, accompanied with a significant increase (P<0.05) in the malondialdehyde level and percentage of abnormal sperm deformity compared with control group. No significant difference was found in the weight of testis, epididymus, prostate, seminal vesicles, testosterone hormone level and body weight compared with control group. The treatment of adult male rats with royal jelly concomitantly with hydrogen peroxide caused a significant increase (P<0.05) in testicular weight and the body of epididymus, sperm count, testosterone hormone and glutathione level, and decrease in sperm deformity percentage, while no significant differences in the prostate weight, seminal vesicles, the percentage of live sperm, malondialdehyde level and body weight compared with hydrogen peroxide group. The treatment of adult male rats with royal jelly alone produced a significant increase (P<0.05) in the weights of testis and body of epididymus, sperm count, testosterone hormone, the percentage of live sperm, and glutathione level and retuned to control value, accompanied with a significant decrease (P<0.05) in malondialdehyde level and the percentage of sperm abnormality. It could be concluded from this study that royal jelly is a beneficial treatment of male adult rats receiving hydrogen peroxide (to induced oxidative stress) specially on sperm count, testosterone hormone level, the percentage of live sperm, and improvement of glutathione and malondialdehyde tissue testis.
This chapter discusses the methods used for the qualitative and quantitative determination of aldehydes in biological systems. It focuses on 4-hydroxynonenal (HNE) and malondialdehyde (MDA). 4-Hydroxynonenal is produced as a major product of the peroxidative decomposition of polyunsaturated fatty acids (PUFA) and possesses cytotoxic, hepatotoxic, mutagenic, and genoroxic properties. Increased levels of HNE are found in plasma and various organs under conditions of oxidative stress. In addition to HNE, lipid peroxidation generates many other aldehydes that may also be of toxicological significance. Malondialdehyde is in many instances the most abundant individual aldehyde resulting from lipid peroxidation, and its determination by thiobarbituric acid (TBA) is one of the most common assays in lipid peroxidation studies. In vitro MDA can alter proteins, DNA, RNA, and many other biomolecules. Recently, it has been demonstrated with monoclonal antibodies that malonaldehyde-altered protein occurs in atheroma of hyperlipidemic rabbits.
OBJECTIVES The study examined arterial and cardiac structure and function in bodybuilders using androgenic anabolic steroids (AAS), compared to non-steroid-using bodybuilder controls.BACKGROUND Adverse cardiovascular events have been reported in bodybuilders taking anabolic steroids. The cardiovascular effects of AAS, however, have not been investigated in detail.METHODS We recruited 20 male bodybuilders (aged 35 ± 3 years), 10 actively using AAS and 10 who denied ever using steroids. Serum lipid and hormone levels, carotid intima-media thickness (IMT), arterial reactivity, and left ventricular (LV) dimensions were measured. Vessel diameter was measured by ultrasound at rest, during reactive hyperemia (an endothelium-dependent response, leading to flow-mediated dilation, FMD), and after sublingual nitroglycerin (GTN, an endothelium-independent dilator). Arterial reactivity was also measured in 10 age-matched non-bodybuilding sedentary controls.RESULTSUse of AAS was associated with significant decreases in high density lipoprotein cholesterol, sex hormone binding globulin, testosterone and gonadotrophin levels, and significant increases in LV mass and self-reported physical strength (p < 0.05). Carotid IMT (0.60 ± 0.04 mm vs. 0.63 ± 0.07 mm), arterial FMD (4.7 ± 1.4% vs. 4.1 ± 0.7%) and GTN responses (11.0 ± 1.9% vs. 14.4 ± 1.7%) were similar in both bodybuilding groups (p > 0.2). The GTN responses were significantly lower and carotid IMT significantly higher in both bodybuilding groups, however, compared with the non-bodybuilding sedentary controls (p = 0.01).CONCLUSIONS Although high-level bodybuilding is associated with impaired vascular reactivity and increased arterial thickening, the use of AAS per se is not associated with significant abnormalities of arterial structure or function.
In order to study a possible immunomodulatory effect of the royal jelly (RJ) secreted by mandibular and hypopharingeal glands of the worker honeybee (Apis mellifera Linne.) we have used a well established rodent model. The CBA mice were given s.c. 0.1 ml of RJ, 7 days before, or immediately after, the immunization with sheep red blood cells (SRBC). The Y59 rats received i.m. 0.4 ml or i.v. 0.025 ml of RJ once or twice at 7 day intervals. Serum levels of total proteins and immunoglobulins in the rats that received RJ once or twice within a 2-week-period were significantly lower (P ≤ 0.05) as compared with the nontreated animals. In mice which were immunized with 4 x 108 of SRBC 7 days after the application of RJ the number of plaque forming splenocytes was significantly higher (P ≤ 0.05) than that in the controls. Both the weight of inguinal lymph node and the number of peripheral blood lymphocytes were increased (P ≤ 0.05) in RJ-treated mice 3 or 5 days after the immunization, respectively. Neutrophils were decreased (P ≤ 0.05) in the mice that were killed 5 or 10 days after the RJ treatment. Overall these results indicate that RJ exhibited immunomodulatory properties by stimulating antibody production and immunocompetent cell proliferation in mice or depressing humoral immune functions in rats. Both phenomena, though species-related in this model, could probably be reversed by changing the dose or the route of RJ application.
SUMMARYA description is given of a family of test-frames for obtaining an unbiased estimate of the numerical density of arbitrary profiles on a section. The counting rule pertaining to the test-frame is simple and requires no corrections based on other estimated quantities.
Royal jelly peptides (RJPx) isolated from hydrolysates of water-soluble royal jelly proteins prepared with protease P exhibited significantly stronger hydroxyl radical-scavenging activity (p<0.001), and antioxidant activity against lipid peroxidation (LPO, p<0.001), than did water-soluble royal jelly protein (WSRJP) in vitro. We also investigated the in vivo antioxidant activity of RJPx against ferric nitrilotriacetate (Fe-NTA)-induced LPO. Male Wistar rats were divided into a control group (Group C), an Fe-NTA group (Group Fe), and an Fe-NTA with RJPx group (Group Fe+R). Rats in Group Fe+R were fed RJPx (2 g/kg body weight) daily for 5 wk. Fe-NTA (8 mg Fe/kg body weight) was then intraperitoneally injected, and serum lipid levels were examined 2 h later. Serum total cholesterol (TC) levels were lower (p<0.05) while low-density lipoprotein (LDL) and LPO were significantly higher (p<0.01) in Group Fe than in Group C. TC (p<0.05) and LPO levels (p<0.01) were lower in Group Fe+R than in Group Fe. Our data suggest that RJPx may inhibit LPO both in vitro and in vivo.
This study was conducted to examine the efficacy of ghrelin in prevention of deleterious effects of heat stress in rat testicular tissue. Forty five adult male rats were scheduled for this study and were divided equally into three groups: heat-saline, heat-ghrelin and control-saline. The scrota of heated-designed rats were immersed once in water bath at 43 °C for 15 min. Immediately upon heating, 2 nmol of ghrelin were given subcutaneously to heat-ghrelin animals every other day up to day 60 and physiological saline to the other two groups using the same method. The animals were sacrificed at 10, 30 and 60 days after heat treatment and their testes were taken for later photomicrograph and immunohistochemical analysis. Testicular histopathology revealed a significant reduction in the means of seminiferous tubules and Sertoli cell nucleus diameters as well as germinal epithelium height on day 10 in both heated groups. Furthermore, other testicular components including miotic index, spermatogenesis rate, presence of spermatocytes and volume densities were dramatically decreased following heat exposure. Notably, ghrelin caused a partial recovery in all of the above-mentioned parameters and accelerated testicular regeneration process by day 30 compared to the heat-saline group (P<0.05). Because of testicular progressive recovery, these indices were similar among groups on day 60 (P>0.05). However, immunohistochemistry evaluation for in situ detection of Bcl-2 protein did not exhibit any germ cells-positive of this factor among groups at different experimental days. In conclusion, the results of the present study indicate for the first time the novel evidences of ghrelin ability in attenuation of heat-induced testicular damage and also that ghrelin therapy may be useful as a suppressor of degenerative effects following testicular hyperthermia.