Influence of royal jelly on the reproductive function of puberty male rats
The adverse effects of royal jelly on the reproductive system of puberty male rats were investigated. Royal jelly was daily administered by gavage to Sprague-Dawley rats at doses 200, 400, and 800 mg/kg for 4 weeks. The body weight and organ coefficients were determined. Sperm count, spermatozoa abnormality, and testicular histopathology were examined through light microscopy. Radioimmunoassay was used to detect serum hormones. The dietary exposure to royal jelly did not affect body weight, but the organ coefficients for the pituitary and testis in the high-dose group were decreased significantly compared with the control group, and significant changes in the microstructure of the testis were observed. No significant differences in sperm count were observed among all groups, however, the sperm deformity rate in the high-dose group increased significantly. Serum hormones in the high-dose group were significantly different from the control group. After royal jelly was stopped for 14 days, the adverse changes were partially reversed and returned to levels close to those in the control group. In conclusion, high-dose royal jelly oral administration for 4 weeks adversely affected the reproductive system of pubescent male rats, but the unfavorable effects are alleviated to some extent by cessation of administration.
Inﬂuence of royal jelly on the reproductive function of puberty male rats
, Ming Zhou
, Li Zhang
, Guoxiu Xie
, Hongbing Chen
, Zhiyong Liu
, Wei Ma
State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang 330047, China
Experimental Animal Center, Jiangxi Institute of Occupational Disease Prevention, Nanchang 330006, China
Supervision and Test Center (Nanchang) for Quality and Safety of Agricultural Products, Ministry of Agriculture, Nanchang 330047, China
Received 3 November 2011
Accepted 29 February 2012
Available online 8 March 2012
Pubescent male rats
The adverse effects of royal jelly on the reproductive system of puberty male rats were investigated. Royal
jelly was daily administered by gavage to Sprague–Dawley rats at doses 200, 400, and 800 mg/kg for
4 weeks. The body weight and organ coefﬁcients were determined. Sperm count, spermatozoa abnormal-
ity, and testicular histopathology were examined through light microscopy. Radioimmunoassay was used
to detect serum hormones. The dietary exposure to royal jelly did not affect body weight, but the organ
coefﬁcients for the pituitary and testis in the high-dose group were decreased signiﬁcantly compared
with the control group, and signiﬁcant changes in the microstructure of the testis were observed. No sig-
niﬁcant differences in sperm count were observed among all groups, however, the sperm deformity rate
in the high-dose group increased signiﬁcantly. Serum hormones in the high-dose group were signiﬁcantly
different from the control group. After royal jelly was stopped for 14 days, the adverse changes were par-
tially reversed and returned to levels close to those in the control group. In conclusion, high-dose royal
jelly oral administration for 4 weeks adversely affected the reproductive system of pubescent male rats,
but the unfavorable effects are alleviated to some extent by cessation of administration.
Ó 2012 Elsevier Ltd. All rights reserved.
Royal jelly, the principal food source of the queen honeybee, is
secreted by the hypopharyngeal glands of nurse bees, and it con-
tains many important nutritious constituents, such as proteins,
sugars, free amino acids, fatty acids, minerals, and vitamins
(Vucevic et al., 2007). Royal jelly possesses many physical and
chemical properties, including anti-inﬂammatory, antioxidant,
anti-tumor, and immunomodulatory functions in experimental
animals (Hattori et al., 2007), which are beneﬁcial to human
health, leading to its wide use in commercial and medical products,
health food, and cosmetics (Guo et al., 2009). Clinical studies have
demonstrated that royal jelly can alleviate osteoporosis and men-
opausal symptoms (Hidaka et al., 2006). Some ﬁndings suggest
that royal jelly has estrogenic activity similar to other exogenous
steroid hormones (Nakaya et al., 2007; Hidaka et al., 2006).
Exogenous estrogen or estrogen-like compounds can be present
in many substances such as seeds, vegetables, milk, and dairy
products (Anniea et al., 2006; Lee et al., 2010).
Both females and males secrete estrogens, which have been
shown to be indispensable for the reproductive system. Estrogenic
compounds have been reported to appear similar to mammalian
estrogens, both structurally and functionally, and it binds to estro-
gen receptors (ERs), which are highly concentrated in the repro-
ductive tract than in the other tissues. Estrogenic compounds
may exert various estrogenic or anti-estrogenic effects in the
reproductive systems by modulating the ERs, making the male
and female reproductive organs, including testes and uterus, vul-
nerable (Beckera et al., 2011; Ceccarelli et al., 2009).
Over the past few years, a decline in semen quantity and quality
has been observed among young men. Researchers have warned
that an increase in male reproductive tract disorders, such as dys-
spermia and testicular infertility, stemmed from increasing expo-
sure of developing males to environmental estrogens (Olesen
et al., 2007). Currently, environmental exogenous estrogens, which
may affect the fertility of male reproductive system in human and
wildlife populations by their interference with estrogen and andro-
gen signaling pathways, have received great attention.
Numerous reports suggest that exposure of male fetuses to
estrogenic compounds may be responsible for reproductive deﬁ-
ciencies in adult life (Sultan et al., 2001). If the estrogen exposure
continued during the prenatal period, a reduction in the weight
of the adult testes was observed (Wisniewski et al., 2005). Marked
atrophy of the testes is caused by neonatal exposure to the
0278-6915/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
Abbreviations: APES, 3-aminopropyl-triethoxysilane; E2, estradiol; ERs, estrogen
receptors; FSH, follicle-stimulating hormone; LH, luteinizing hormone; PBS, phos-
phate-buffered saline; Ts, testosterone.
Corresponding author. Tel.: +86 791 88629471; fax: +86 791 8860957.
E-mail addresses: email@example.com, firstname.lastname@example.org
Food and Chemical Toxicology 50 (2012) 1834–1840
Contents lists available at SciVerse ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox
exogenous hormone, diethylstilbestrol (Adachi et al., 2003). The
sexual behavior of male rats is sensitive to phytoestrogen expo-
sure. Studies have shown that phytoestrogens are present in the
environment at concentrations high enough to have long-term ad-
verse effects on males at adulthood and alter their reproductive
function through estrogenic or anti-androgenic activities (Santti
et al., 1998). It has been demonstrated that exposure to estrogen
during early, critical periods of development is associated with
endocrine system and reproductive tract alterations (Herbst
et al., 1972; McLachlan et al., 1980).
It has been reported that orally exposure to royal jelly exerted
estrogenic effects in adult female rats. For ewes, royal jelly may
be effective in improving pregnancy and lambing rates (Kridli
and Al-Khetib, 2006). For males, Nakaya et al. (2007) found that
royal jelly has anti-environmental estrogen activity, which can in-
hibit the negative effects exerted by exogenous estrogen on male
reproductive status. In the same way, Abdelhaﬁz and Muhamad
(2008) reported that the administration of Egyptian bee honey
and royal jelly intravaginally might be a reasonably effective meth-
od for treating infertility caused by asthenozoospermia. Previous
studies show that royal jelly possesses many health beneﬁts. How-
ever, little information is available on the effects of royal jelly on
the reproductive status of male animals. Additional research on
the treatment dose, method or timing of administration, and the
effects of the mechanism should be conducted. In the current
study, the oral administration of royal jelly was evaluated for any
effects on the development of sexual organs and reproductive per-
formance of pubescent male rats, including body weight, organ
coefﬁcients, total sperm count, spermatozoa abnormality, histopa-
thology of testis and sexual hormones.
2. Material and methods
Puberty male Sprague–Dawley rats were obtained from Jiangxi Laboratory Ani-
mal Center (Nanchang, China). All rats were allowed access to water and food ad
libitum under the controlled conditions of temperature (22±2 °C), humidity
(55 ± 5%) and light (12 and 12 h of light and dark, respectively). Two rats were
housed in a plastic cage containing sterile paddy husk (procured locally) as bedding
throughout the experiment. Fresh royal jelly was purchased from Jiangxi Agricul-
tural University (Nanchang, China).
2.2. Animal experimental design
Royal jelly secreted by the hypopharyngeal glands of nurse bees was dissolved
in water to prepare a concentration equivalent to the experimental doses. Thirty-
two Sprague–Dawley male rats (45 days old, 170 ± 10 g) were randomly divided
equally into four groups. All groups were fed normally with rat feed and water,
but the three groups rats were gavaged with the low (200 mg/kg), moderate
(400 mg/kg), and high (800 mg/kg) doses of royal jelly, respectively, every day. Body
weight was measured and recorded weekly. The general physical condition of each
rat was observed daily. After 4 weeks, the oral administration was stopped, half of
rats for each group were killed, arterial blood was collected, and serum was ob-
tained via centrifugation and collected for hormone assay. The bodies were dis-
sected and some organs (liver, kidney, spleen, brain, pituitary, prostate, seminal
vesicle, testis and epididymis) were removed for biochemical analysis. The remain-
ing rats were subsequently fed normally without royal jelly for 14 days, after which
they were killed. The same operations were carried out as above.
2.3. Body weight and organ coefﬁcient
The body weight of the rats was measured every week, and the weight growth
curves of the rats during the period were described. After the rats were killed, their
liver, spleen, kidneys, pituitary gland, prostate gland, seminal vesicles, epididymis,
and testicle were excised and weighed to calculate the organ coefﬁcient (organ/
body weight ratios) for each rat.
2.4. Histopathologic observation of testicles
After the rats were killed, the fresh testicular tissues were harvested and ﬁxed
in aqueous Bouin’s ﬂuid for 24 h. The tissues were then dehydrated in ethanol,
cleared in xylene, and embedded in parafﬁn at 58 °C. The embedded tissue samples
were cut into 5
m thick sections using a rotary microtome, pretreated with stock
APES solution, and dried in an oven at 50 °C for 12 h. After deparafﬁnization by
immersion in xylene for 20 min, the tissues were dropped in ethanol solutions with
decreasing concentrations (100%, 95%, 90%, and 80%), respectively, for 5 min. The
sections were then stained with hematoxylin and eosin. Histopathology in the testis
were examined through a microscope. Micrographs were taken to observe the mor-
phology of any lesions (400).
2.5. Evaluation of sperm count and sperm morphology
To analyze the effects of exposure to royal jelly on rat spermatogenesis, the cau-
da epididymides were immediately dissected and weighed. The epididymides were
then minced and homogenized for 10 s in 5.0 ml of physiologic saline solution. The
resulting sperm suspension was centrifuged at 1000 rpm for 10 s. Up to 200
the supernate was then incubated with 800
l of PBS. Half volume of it was used
for counting. Fifteen micro liters of the dilution of sperm suspension was placed
on a hemocytometer to count the number of sperms under light microscopy at
400 magniﬁcation. The average sperm number was counted in triplicate. To eval-
uate the sperm morphology, the remaining half of the cauda epididymis was incu-
bated at room temperature in 0.9% saline, and the sperm cells were allowed to
disperse for 5 min. After gentle agitation, 200
l of the sperm suspension was
smeared onto a slide glass and stained with 2% eosin for 1 h (Trivedi et al., 2010).
Two hundred and ﬁfty sperms from each sample were classiﬁed as normal, with
head defect, and with tail defect to calculating the percentage of abnormal sperm.
2.6. Hormone determinations
Serum hormones, namely, follicle-stimulating hormone (FSH), luteinizing hor-
mone (LH), estradiol (E2), and testosterone (Ts), were detected by radioimmunoas-
say according to the procedures provided by the manufacturer’s instructions. Intra
and inter-assay coefﬁcients of variation, respectively, were below or equal to 11.5%
and 14.4% for FSH, 9.4% and 11.2% for LH, 6.1% and 9.4% for E2, and 3.3% and 6.9% for
2.7. Statistical analysis
All the experiments were repeated three times and the results were expressed
as mean ± S.D. Statistical analysis was done using two-tailed Student’s t-test. In all
cases, P < 0.05 was considered to be signiﬁcant.
3.1. Changes in body weight and main organ coefﬁcients of rats
The effects of royal jelly on rat body weight are shown in Fig. 1.
The body weight of male rats increased gradually by week until
they were killed. The body weights of male rats exposed to royal
jelly at any dose were not signiﬁcantly different from those of
the control group throughout the experimental period. Royal jelly
did not impart a signiﬁcant effect on the body weight of the male
Body weight (g)
Treatment time (weeks)
Fig. 1. Body weight of rats exposed to royal jelly in different weeks.
A. Yang et al. / Food and Chemical Toxicology 50 (2012) 1834–1840
To determine the target organ that interacts with royal jelly, the
main organ coefﬁcient of rats by oral gavage was evaluated. Table 1
shows that the male rats fed with royal jelly exhibited a dose-
dependent increase in the organ coefﬁcient of the liver. In contrast,
the pituitary organ coefﬁcient of the rats exposed to royal jelly at
any dose was decreased and that of the high-dose group showed
signiﬁcant differences compared with the control group
(P < 0.05), whereas the other normal organ coefﬁcients of rats, such
as kidney, spleen, and brain, were not affected by the dose of royal
The inﬂuence of royal jelly on the reproductive organ coefﬁcient
was also explored. Compared with the control group, the organ
coefﬁcient for the testis in the high-dose group was signiﬁcantly
decreased. However, the organ coefﬁcient for the prostate in all
groups was increased and it is signiﬁcantly different from that of
the low-dose group (P < 0.05), whereas the organ coefﬁcient for
the seminal vesicles in the moderate-dose group was signiﬁcantly
higher than that of the control group (P < 0.05).
After royal jelly treatment and 14 days without royal jelly, as
indicated in Table 2, only the organ coefﬁcient for the testis in
the high-dose group was signiﬁcantly different from that of the
control group (P<0.05). No signiﬁcant differences were observed
in the other organ coefﬁcients of the rats exposed to royal jelly
at any dose compared with the control group.
3.2. Testicular morphology and histopathology
The histopathology of the testis in all royal jelly-treated groups
was observed through light microscopic examinations (Fig. 2). The
testis in the control group was pink, smooth, and plump with
abundant seminiferous tubules. The basal lamina was clear and in-
tact. Approximately ﬁve or six layers of seminiferous epithelia
were present, and the arrangements of the spermatogenic cells
and Sertoli cells were in good order. As for the administration
groups, the numbers of spermatogenic epithelium in rats testicular
were decreased with the increase of royal jelly-treated dose. At
high dose group, degeneration of seminiferous tubules was evident
with necrotic cellular debris found in some tubules, and multi-
nucleaed giant cells appeared within the individual lumen. A
reduction in the number of Leydig cells around the seminiferous
tubule was observed. The cytoplasm of the Leydig cells was con-
densed, and the nuclei were hyperchromatic and surrounded by
larger vacuoles. However, neither signiﬁcant inﬂammatory nor
bleeding was found in interstitial tissue. Continuous high-dose
exposure to royal jelly might have certain degree of adverse effect
on the microstructure of rat testes. After 14 days without royal jel-
ly, seminiferous epithelia in each dosage group were gradually
recovered. The visible layers were increased, and the different per-
iod of sperms could be observed, especially in the high dose.
3.3. Sperm count and morphology
At the end of the 28-day royal jelly treatment period, the sperm
count and percentage of abnormal sperm were determined (Table
3). No signiﬁcant changes in the number of sperm were recorded in
all treatment groups compared with the control. However, royal
jelly exposure affected the sperm morphology in the three groups
with different doses of royal jelly. Signiﬁcant increases were ob-
served in the abnormal sperm percentages of moderate and high
royal jelly treatment groups compared with the control group,
and the head defects were observed as the predominant type of
sperm malformation in comparison with tail defects. Head defects
include double-headed, without hook and amorphous, and tail de-
fects are divided into double tail and tail folding (Fig. 3). After
14 days without royal jelly, no great changes in the count of sperm
were observed, and sperm deformity rate were decreased in all
treatment groups. However, the abnormal sperm percentages in
high royal jelly treatment group was still signiﬁcant higher than
that of the control group (Table 3).
3.4. Serum hormone concentration
Exogenous estrogen interacted with the ERs through which it
might have affected the serum hormone levels by modulating hor-
mone secretion and expression. Based on the results above, the ser-
um levels of sexual hormone, such as FSH, LH, E2, and Ts, were
detected. As shown in Fig. 4, the serum FSH levels of the rats ex-
posed to royal jelly at all doses were decreased compared with
the controls, and the higher the royal jelly dose, the lower the
FSH level was detected in serum of the rats. The changes in serum
LH levels between the high-dose group and the control group were
signiﬁcant (P < 0.05). However, the LH levels in the low- and mod-
erate-dose groups did not differ from those of the high-dose group,
but were close to the control group. For E2, no signiﬁcant changes
were observed in the low and moderate-dose groups orally given
royal jelly, whereas the E2 content in the high-dose group were in-
creased signiﬁcantly compared with the control group (
P < 0.05).
The serum Ts levels in the high-dose group were close to the mod-
erate-dose group and signiﬁcantly decreased compared with the
control group (P < 0.05). After 14 days without royal jelly, the ser-
um levels of FSH, LH, and Ts in the high-dose group were increased
signiﬁcantly and approached those of the control group. In con-
trast, the E2 level in the high-dose group was decreased distinctly,
with values similar to those of the control group.
In recent years, environmental estrogens have aroused concern
in both public and scientiﬁc communities. Exposure of wildlife and
Organ coefﬁcients (g/100 g body weight) in puberty male rats orally given royal jelly.
Organ Royal jelly (mg/kg)
Control Low (200) Moderate
Liver 2.60 ± 0.20 2.67 ± 0.14 2.74 ± 0.16 2.89 ± 0.22
Kidney 0.71 ± 0.04 0.75 ± 0.03 0.72 ± 0.07 0.75 ± 0.20
Spleen 0.20 ± 0.05 0.17 ± 0.01 0.19 ± 0.01 0.19 ± 0.02
Brain 0.41 ± 0.01 0.38 ± 0.04 0.39 ± 0.01 0.41 ± 0.01
) 0.38 ± 0.04 0.33 ± 0.02 0.34 ± 0.04 0.26 ± 0.03⁄
Prostate 0.10 ± 0.05 0.22 ± 0.11
0.16 ± 0.03 0.17 ± 0.02
Seminal vesicle 0.24 ± 0.04 0.21 ± 0.05 0.32 ± 0.01
0.28 ± 0.02
Testis 0.85 ± 0.07 0.77 ± 0.05 0.77 ± 0.07 0.76 ± 0.03
Epididymis 0.16 ± 0.02 0.17 ± 0.02 0.14 ± 0.02 0.15 ± 0.02
P<0.05 vs. control.
Organ coefﬁcients (g/100 g body weight) of puberty male rats after royal jelly
treatment followed by 14 days without royal jelly.
Organ Royal jelly (mg/kg)
Control Low (200) Moderate (400) High (800)
Liver 3.23 ± 0.27 3.39 ± 0.24 3.36 ± 0.20 3.15 ± 0.25
Kidney 0.62 ± 0.04 0.65 ± 0.31 0.69 ± 0.71 0.65 ± 0.54
Spleen 0.19 ± 0.02 0.18 ± 0.03 0.18 ± 0.02 0.17 ± 0.01
Brain 0.33 ± 0.02 0.32 ± 0.02 0.32 ± 0.02 0.30 ± 0.01
) 0.35 ± 0.05 0.38 ± 0.07 0.29 ± 0.03 0.30 ± 0.04
Prostate 0.17 ± 0.01 0.18 ± 0.05 0.17 ± 0.02 0.18 ± 0.01
Seminal vesicle 0.32 ± 0.02 0.33 ± 0.04 0.26 ± 0.08 0.30 ± 0.06
Testis 0.66 ± 0.02 0.63 ± 0.03 0.62 ± 0.05 0.59 ± 0.02
Epididymis 0.12 ± 0.01 0.13 ± 0.02 0.10 ± 0.02 0.13 ± 0.02
P<0.05 vs. control.
1836 A. Yang et al. / Food and Chemical Toxicology 50 (2012) 1834–1840
humans to exogenous estrogen may be responsible for disorders of
the reproductive system such as cryptorchidism, dysspermia, and
testicular structural anomalies. Royal jelly has estrogenic activity,
as demonstrated by some previous studies (Mishima et al., 2005).
Similar to other exogenous estrogens, royal jelly elicits estrogen-
like effects through interaction with ERs, which may lead to
Sperm count and sperm deformity rate in the cauda epididymides of male rats exposed to royal jelly (I) and after 14 days without royal jelly (II).
Time Group Sperm count (10
/ml) Sperm deformity rate (%) Head defects Tail defects
I Control 41.43 ± 4.00 3.01 ± 0.31 2.70 ± 0.32 0.32 ± 0.07
Low 38.31 ± 4.46 3.45 ± 0.34 2.74 ± 0.22 0.71 ± 0.05
Moderate 38.09 ± 2.16 4.98 ± 0.41
4.43 ± 0.53 0.56 ± 0.04
High 39.82 ± 3.12 6.16 ± 0.52
5.42 ± 0.41 0.73 ± 0.04
II Control 42.10 ± 4.36 3.12 ± 0.29 2.59 ± 0.27 0.35 ± 0.06
Low 41.02 ± 4.62 3.22 ± 0.40 2.67 ± 0.29 0.64 ± 0.04
Moderate 40.57 ± 3.93 4.07 ± 0.47 3.31 ± 0.38 0.63 ± 0.05
High 40.10 ± 4.05 5.25 ± 0.42
4.67 ± 0.40 0.75 ± 0.04
P<0.05 vs. control.
Fig. 2. Histopathology of the testis in pubescent male rats exposed to royal jelly (A: control, B: low dose, C: moderate dose, D: high dose) and after 14 days without royal jelly
(E: control, F: low dose, G: moderate dose, H: high dose) with different dose.
A. Yang et al. / Food and Chemical Toxicology 50 (2012) 1834–1840
changes in male reproductive capability. Elnagar reported that at
proper levels, royal jelly has a positive effect on animal reproduc-
tive status (Elnagar, 2010).
Recently, great attention has been focused on the safety of royal
jelly. However, the effects of high-dose royal jelly on the normal
pubescent male reproductive system are still unclear. In the pres-
ent study, we studied the possible effects of royal jelly on the
reproductive system of pubescent male rats including the main or-
gan coefﬁcients, total sperm count, spermatozoa abnormality, his-
topathology of testis, and sexual hormone level.
In toxicological studies, organ coefﬁcients are important criteria
for evaluating organ toxicity. In the current study, no statistically
signiﬁcant difference in body weight was found and most of organ
coefﬁcients of male rats treated with royal jelly in the low- and
moderate-dose groups compared with control group. However,
the pituitary and testis organ coefﬁcients for the rats in the high-
dose group decreased signiﬁcantly (P < 0.05).
In males, the secretion of the two pituitary hormones (gonado-
tropins), namely, LH and FSH is stimulated by gonadotropin-releas-
ing hormone derived from hypothalamus, and LH, FSH, as well as
testosterone secreted from the testis play important roles in the
initiation and early stages of spermatogenic growth (Zhou et al.,
2008). LH stimulates Leydig cells via its receptors to synthesize
and secrete testosterone, whereas FSH acts on the Sertoli cells in
the seminiferous epithelium through speciﬁc receptors and pro-
motes spermatogenesis. According to their different developmen-
tal stages, spermatogenic cel1 included spermatogonia, primary
spermatocytes, secondary spermatocytes, spermatids and sperm.
The continuous proliferation differentiation process within from
spermatogonia to sperm formation was called spermatogenesis.
Testosterone secretion and spermatogenesis support of male sec-
ondary sexual characteristics greatly inﬂuence the development
of the immature testis.
Several physiologic studies suggest that male sexual differenti-
ation and reproductive functions are controlled by the regulation
of various hormones, and hormonal regulation begins with fetus
and continues through puberty and into adulthood (McLachlan,
2000). However, exposure to exogenous estrogen may disrupt
the balance of various hormones by interference with hypothala-
mus–pituitary–testis axis, and disorder in any part of the system
will disrupt the hormonal balance and harm male reproductive
Similar to the endogenous estrogens, exogenous estrogens can
also interact with ERs and act as endocrine disruptors. Male repro-
ductive disorders may be correlated with environmental exposure
to chemical compounds with hormone-like properties, which can
exert either estrogenic or anti-estrogenic effects. For example,
exogenous estrogens can interfere with the endogenous estrogen
pathways, affecting the production of sex steroid-binding proteins
(Santti et al., 1998). The expression of estrogen and androgen
receptors in the testis is altered at puberty by neonatal exposure
to various exogenous estrogens (Adachi et al., 2004). An estrogen
excess can decrease testicular androgen production by lowering
the activity of steroidogenic enzymes that convert progesterone
to testosterone (Loomis and Thomas, 2000). Thus, exogenous estro-
gen exposure disrupts male reproductive aspects, such as reduced
testicular size and infertility.
In the current study, the histopathology of the testis was abnor-
mal in the high-dose group. Similarly, sperm morphology was
inﬂuenced by high-dose royal jelly exposure, whereas the sperms
count did not show differences among the groups. Thus, high-dose
royal jelly exposure is signiﬁcantly associated with poor sperm
count and increased risk of male infertility. The FSH and LH serum
levels in the rats in the middle- and high-dose royal jelly groups
were signiﬁcantly decreased compared with controls (P < 0.05).
However, the E2 serum levels showed a dose-dependent increase.
Together with the organ coefﬁcient results, long-term exposure to
royal jelly may cause pituitary abnormalities, and reduced FSH
secretion, as well as decreased testosterone secretion and testicu-
lar atrophy. The upregulation of the conversion of testosterone into
E2 also seems to contribute to the decline in plasma testosterone
levels. The signiﬁcant change in testosterone level might be a plau-
sible explanation for the increase in the number of abnormal
sperm in the royal jelly-treated male rats, which is inconsistent
with the ﬁndings of Karacal et al. who reported higher sperm
motility when male mice were treated with royal jelly (Karacal
and Aral, 2008). The contradiction in estrogenic effects of royal jel-
ly on reproductive functions is possibly due to the differences in
the treatment dose, method, or timing of administration, sexuality
of test animal, and the presence of royal jelly composition. After
14 days without royal jelly, the sexual hormone levels were par-
tially restored, which contrasts with the results reported by Guan
et al. who demonstrated that prepubertal exposure to high doses
of soybean isoﬂavone extracts reduces serum testosterone levels
Fig. 3. Photomicrographs on morphology of abnormal sperm (A: head defects with double-headed, B: head defects without hook, C: tail defects with double tail, D: tail
defects with folding).
1838 A. Yang et al. / Food and Chemical Toxicology 50 (2012) 1834–1840
and such changes might persist into adulthood (Guan et al., 2008).
The results conﬁrmed the afﬁnity of royal jelly for ERs were weaker
than those of phytoestrogens (Routledge et al., 2000). Therefore,
the adverse changes of the reproductive system in puberty male
rats were partially inhibited after some time without royal jelly.
In conclusion, the current study shows no signiﬁcant alterations
in body weight, and the organ coefﬁcients for the pituitary and tes-
tis in the high-dose group were signiﬁcantly decreased compared
with the control group. Continued exposure of pubescent male rats
to royal jelly at high doses level damages the testicular microstruc-
ture, the development of spermatogenesis, and disrupts the
homeostasis of reproductive hormones, but the change is partially
reversed by 14 days without royal jelly. The reproductive system of
pubescent male rats is relatively sensitive to royal jelly and their
reproductive function is likely to be affected by high-dose admin-
istration for 4 weeks, however, spontaneous recovery occurs after
royal jelly is stopped if the extent of damage is much lower than
the threshold limit. Further research is in progress to determine
the mechanisms underlying the effects of royal jelly in pubescent
Conﬂict of Interest
The authors declare that there are no conﬂicts of interest.
This work was supported by Science and Technology Support
Program of Jiangxi Province (Project No. 20111BBF60025), Re-
search Foundation for Young Scientists of State Key Laboratory of
Food Science and Technology (Project No. SKLF-QN-201111), Open
Project Program (Project No. SKLF-KF-201007) and Research Pro-
gram (Project No. SKLF-TS-201109, SKLF-MB-201002) of State
Key Laboratory of Food Science and Technology, Nanchang
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