Effects of lepidium sativum (garden cress) seed on plasma luteinizing hormone and reproductive performance in female rabbits

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J. Afr. Ass. Physiol. Sci. 6 (1): 79-84, July 2018
Journal of African Association of Physiological Sciences
Official Publication of the African Association of Physiological Sciences
http://www.jaaps.aapsnet.org
Research Article
Effects of dietary inclusion of Lepidium sativum (garden cress)
seed on plasma luteinizing hormone and reproductive
performance in female rabbits
*1,3O.V. Imade, O.F. Smith1 and O.S. Gazal2
1Department of Animal Physiology, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria., 2Animal
Reproduction and Endocrinology Laboratory, Department of Biological Sciences, College of Science and
Engineering, St. Cloud State University, St. Cloud, Minnesota 56301, USA. and 3Sciences Department, Faculty of
Education, Kampala International University, Western Campus, Ishaka, Bushenyi District, Uganda.
Keywords:
Lepidium sativum seed,
rabbit, reproductive
performance, luteinizing
hormone, pregnancy,
foetus
,
ABSTRACT
Background: Lepidium sativum (LS) has been reported to have multi-purpose medicinal uses.
For instance, rubefacient, antihypertensive, hepatoprotective, galactagogue and aphrodisiac
properties are well documented; however, there are few controversial reports particularly as
related to reproduction. Also, no known study has identified the mechanisms underlying the
action of this plant on reproduction. This study was aimed at evaluating the effects of dietary
supplementation with LS on luteinizing hormone and reproductive performance in female
rabbits. Methods: A total of twenty, nulliparous Chinchilla female rabbits (6.5-month old)
were randomly assigned to four treatment groups with five (n=5) rabbits in each group. Diet
containing either LS seed powder at 0% (Control), 5%, 7% or 10% were given ad-libitum to
female rabbits two weeks pre-pregnancy and throughout the entire period of pregnancy. Blood
samples were collected through the marginal ear vein from each female rabbit weekly during
pre-pregnancy and pregnancy periods to evaluate Luteinizing hormone (LH). Conception rate,
gestation length, litter size, litter weight, number of stillbirths and runts were recorded. Data
obtained were analyzed using ANOVA. Results: LS seed inclusion significantly increased
(P<0.05) conception rate and plasma LH concentration in dose dependent manner, from 5%- to
the 7%- and then decreased at 10%-LS seed level. There was a significant decrease (P<0.05) in
gestation length in does fed 10% LS-based diet. Furthermore, the litter weights and number of
live kid born were significantly (P<0.05) decreased in all LS groups. In contrast to zero
incidence in control rabbits, high incidence of stillbirth and runt were recorded in all LS-based
groups. Conclusion: The results show that inclusion of LS seed in rabbit chow increases
conception rate and plasma LH levels which were attributed to the phytoestrogens constituent
in the seed. Despite the positive role of LS seeds on conception rate and LH, LS seed may
possess fetal developmental toxicity effects due to high incidence of stillbirth and extremely
low birth weight of newborn recorded.
© Copyright 2018 African Association of Physiological Sciences -ISSN: 2315-9987. All rights reserved
INTRODUCTION
For many years, various ethnic communities understand
and have secret ethnomedicinal knowledge about many
plants available in their environments and these plants
have been serving them with their advantages (Taid et
al., 2014). The biological activities of the medicinal
plants have been accredited to chemical constituents
that induce or suppress particular physiological action
in the body system (Faraz et al., 2003).
Though, lot of the acclaimed properties and
physiological actions of plants used in traditional
system of medicine are not scientifically defined
especially those ones related to reproduction.
Lepidium sativum (LS), also known as garden cress in
English, Jan Algaro or Lansir in Hausa (Northern
Nigeria), Habburshad (Arabian), Chandrasur (Hindi),
Lepido (Spanish) and Cresson de fontaine in French is
been reported to possess various multi-system effects in
traditional medicine, such as anti-rheumatism (Ahsan et
al., 1989); anti-diarrhoea (Rahimi et al., 2010); anti-
febrifuge (Ageel et al., 1987); rubefacient and
galactagogue (Kirtikar and Basu, 1995) etc. Potentials
*Address for correspondence:
Email tosinimade@gmail.com
Tel: +234 7035760320

Lepidium sativum and pregnancy outcome in rabbits
J. Afr. Ass. Physiol. Sci. 6 (1): 2018 Imade et al.
of LS as related to reproduction were also documented.
For instance, LS seeds roasted and mixed with sugar
are given as an effective medicine to increase breast
milk (Pullaiah, 2006). It’s also reported to increase
sexual stamina and sexual retentivity (Duke, 1992;
Mali et al., 2007; Jabeen et al., 2017). The plant is
considered as abortifacient in human (Baquar, 1989;
Duke, 1992). It is also recorded to possess teratogenic
(Nath et al., 1992), antifertility (Falana et al., 2014) and
antiovulatory effects (Satyavati, 1984). As a result of
the aforementioned reproductive properties of LS, some
scientific studies were carried out on the various parts
of the plant. Supplementation of LS seed powder into
the feed of lactating buffaloes increases their total milk
yield (Abo El-Nor et al., 2007; Kumar et al., 2011). A
mammogenic and lactogenic activity of LS was
reported in rats administered 1.6mg/gm BW of LS seed
powder for 14 days (Al-Yawer et al., 2006). Moreover,
proceptivity and receptivity property was reported in
ovariectomized rat when 200 and 400 mg/Kg BW of
methanolic extract of LS were given orally for 21 days
(Kagathara et al., 2015). Similarly, it was reported to
increase prolactin, progesterone and luteinizing
hormone in ovariectomized rats (Kagathara et al.,
2015). However, Shareif and Gani, (2004)
reported 100% oral contraceptive effects of L. sativum
seed in female mice.
There is a divergence of opinion on the effects of L.
sativum seed on reproduction. Furthermore,
information on efficacy, safety and mechanism of
action is lacking. Moreover, little is known about it
effects on LH secretion and reproductive performance
in mammals. Hence, this study was undertaken to
investigate the effects of dietary inclusion of Lepidium
sativum seed on plasma LH and reproductive
performance in female rabbits.
MATERIALS AND METHODS
Plant material and preparation
Lepidium sativum (Garden cress) seeds were purchased
from a farmer at local market, Gombe, Gombe State,
Nigeria. The LS seeds were identified and
authenticated in the Herbarium of the Department of
Forestry and Wildlife Management, Federal University
of Agriculture, Abeokuta, Nigeria by Dr. J.A. Soaga.
The seeds were ground into fine powder
using electric mill and kept in an airtight container for
later use.
Experimental animals
Twenty (20) nulliparous Chinchilla rabbits (mean
weight, 2.05 ± 0.1 Kg; 6.5 months of age) were
obtained from a reputable breeder farm at Abeokuta,
Ogun State, Nigeria. The rabbits were randomly
assigned into four different groups, animals in group I,
II, III and IV (n=5 per group) were fed with diet
containing Lepidium sativum seed powder at 0%
(Standard rabbit feed, Control), 5%, 7% and 10%,
respectively (w/w). The animals were housed
individually in a wooden cage where feed and water
were given ad libitum. The research was carried out at
the Department of Animal Physiology, Federal
university of Agriculture, Abeokuta, Ogun State,
Nigeria. The animals were housed in a natural open-
sided pen and reared under clean environmental
condition at Favour farms, Abeokuta, Ogun State.
Ethical approval for this study was obtained from the
Research Ethics Committee, College of Animal Science
and Livestock Production, Federal University of
Agriculture, Abeokuta, Ogun State, Nigeria.
Data collection
The animals were given experimental diet for two
weeks before mating and throughout the entire period
of pregnancy. The rabbits were synchronized by
injecting 40 IU (standard approved by The European
Agency for the Evaluation of Medicinal Products)
Pregnant Mare Serum Gonadotropin (PMSG, obtained
from Sigma Aldrich Chemicals, St. Louis, MO, USA)
subcutaneously 72 hours before mating. 72 hours post-
PMSG injection, mating was done naturally by taking
rabbit buck to each doe cage. Mating was visually
observed and all rabbits were receptive. Pregnancy was
tested through palpation after 14 days of mating. Blood
samples were collected from each animal through the
marginal ear vein into lithium heparin tubes once a
week throughout the period of experiment. Blood
samples were centrifuged at 3000 rpm in refrigerated
centrifuge (4 oC) for 15 mins to obtain plasma. Plasma
was stored at -20 oC until LH assay was done using
radioimmunoassay technique. Conception rate,
gestation period, litter size (total, alive and dead),
number of runt, litter weight of the kids at birth and
number of stillbirth were recorded.
Conception rate
Conception was tested at 14-day post mating through
palpation. Conception rate was calculated as a ratio of
the number of does conceived to the total number of
does mated multiplied by 100.
Gestation length and litter size
This was recorded as the interval between mating and
kindling (Oguike and Okocha, 2008). Litter size was
measured by direct counting of newborn immediately
after kindling. It included number of stillbirth (Paci et
al., 2012).
80

Lepidium sativum and pregnancy outcome in rabbits
J. Afr. Ass. Physiol. Sci. 6 (1): 2018 Imade et al.
Litter weight
The newborns were weighed at birth using a sensitive
weighing balance (Oguike and Okocha, 2008). Number
of stillbirths denote the number of dead newborns at
the time of parturition while Number of runt denote
number of newborns with extremely low birth
weight; usually less than average weight of Chinchilla
rabbit at birth (˂ 40g).
LH assay
Plasma LH concentration was determined using
radioimmunoassay technique described by Bernard et
al. (1983). The LH used for the standards and
iodination reaction was obtained from Sigma Aldrich
Chemicals, St. Louis, MO. The antiserum AFP
C5288113 was used at 1:35,000 dilution as the first
antibody and Goat Anti-Rabbit Immunoglobulin G
(GARGG, EQUITECH-BIO INC, Kerrville, Texas,
USA) was used as the second antibody. The samples
were run in duplicates in 3-day assay. The minimum
LH detected by assay was 0.4 ng/mL, and average
intra- and interassay coefficients of variation were
5.1% and 7.4% respectively.
Statistical analysis
Data are presented as Means ± SEM and were
analyzed using statistical program of SAS (2002).
Statistical comparisons were performed by one-way
analysis of variance (ANOVA), followed by Tukey's
multiple comparison test. The main effects of
treatments on plasma LH concentration and
reproductive performances were determined and a p –
value less than 0.05 was considered statistically
significant in all analysis.
RESULTS
Effects of Dietary Inclusion of LS Seed on LH
Concentrations in Female rabbits
Averaged all over sampling periods, dietary inclusion
of LS seed at 5% and 7% significantly (P˂0.05)
increased plasma LH levels compared with the control
or 10% LS inclusion (fig. 1).
Effects of dietary inclusion of lepidium sativum seed on
reproductive performances in female rabbits
Effects of dietary inclusion of Lepidium sativum seed
on reproductive performances in female rabbits are
presented in table 1. The results show a statistically
significant (P˂0.05) higher conception rate in 5% LS
and 7% LS groups (100%) while that of control and
10% LS groups were lower (60%).
Figure 1: Effects of Dietary Inclusion of Lepidium sativum
Seed on Plasma LH Concentrations in Female rabbits. Data
shown mean ± SEM; Bars with asterisks are significantly
different.
The gestation length of control, 5% and 7% LS groups
were not significant (P>0.05) while gestation length in
10% LS group was statistically (P˂0.05) lower
compared with the control and other LS groups. No
significant (P>0.05) difference in the litter size was
observed among the four treatment groups.
Litter weight and number of live newborn of the control
group were significantly (P˂0.05) higher than the LS
groups. Control rabbits recorded neither runt nor
stillbirth. 5% LS group had the highest percentage of
runt (9.1%) with 15.2 % of stillbirth, 7% LS group
recorded 3.6% of runt with 7.1% of stillbirth while 10%
LS group had the highest number of stillbirth with no
runt.
DISCUSSION
In this present study, 5%- and 7%-LS seed inclusion in
the diet of female rabbits increased plasma LH
secretion while a decrease in plasma LH concentration
was observed in 10% LS rabbits. Stimulation of LH
may be ascribed to phytosterol constituent of LS
through temporary or permanent alteration of the
feedback loop in the hypothalamus, pituitary and the
gonad by mimicking the effects of endogenous estrogen
and trigger their specific receptors, thereby resulting in
increased LH secretion. This report is in agreement
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Lepidium sativum and pregnancy outcome in rabbits
J. Afr. Ass. Physiol. Sci. 6 (1): 2018 Imade et al.
Table 1: Effect of Dietary Inclusion of Lepidium sativum
Seed on Reproductive Performance in Female rabbits
a, b, c, d Values in the same row with no common superscript(s)
differ significantly (P<0.05)
with the findings of Al-Yawer et al. (2006) which was
the only known available research work that evaluated
effects of LS on reproduction at the hormonal level.
The authors reported increase in luteinizing hormone in
young adult female virgin rats fed LS supplemented
diet for only 14 days. However, reduction in LH levels
at 10% level of LS inclusion in this present study may
be as a result of adenohypophysial negative feedback of
the estrogenic action of phytosterol, inducing a
decrease or loss of progressive sensitivity or the affinity
for estrogen receptors binding which then causes
subsequent LH inhibition.
The outcome of this study establishes that LS seed
inclusion into the feed of rabbits did not inhibit
ovulation, fertilization or implantation because all the
rabbits were receptive at the time of mating, conception
took place except for lower conception rate recorded in
10% LS group but still within the normal percentage
rate of conception in nulliparous rabbits and the does
carried the pregnancy to term. This is in contrast to the
antiovulatory, contraceptive and abortifacient reports
by Pande et al. (2002); Shareif and Gani, (2004) and
Duke et al. (2002), respectively. The use of different
animal model, administration method, fraction and
dosage of LS seed in these studies could be the reason
for the differences. Previous studies (Duke et al., 2002;
Kagathara et al., 2015) reported receptivity action of
LS seed and this activity may contribute to the high rate
of conception observed in 5 and 7 percent LS groups in
this present study. The receptivity effect may be
ascribed to phytosterols in LS, which mediate their
activity through estrogen receptors by producing
agonistic effects thereby resulting in LH secretion
which is required for the rupture of follicles. The
percentage conception rate of the control group is
within the normal percentage rate of conception in
nulliparous rabbits. The reduction in conception rate in
rabbits fed at the 10 percent LS seed inclusion level
suggests that addition of LS seed at that high level
could alter ovulation and fertility, similar to
documented report by Falana et al. (2014). It is
therefore speculated that this may be more pronounced
at high level of LS seed more than 10%.
LS seed inclusion into the diet of female rabbits
decreased average litter weight and number of live kid
born. The decrease in average litter weight and number
of live kid born in LS groups may be due to maternal
nutrient during pregnancy. Maternal nutrient intake
before and even during pregnancy is critical for foetal
development. Nutrition of the foetus begins at
conception and foetal development may be arrested at
any stage due to many factors including nutrition
(Mertz, 1986). LS seed contain antinutritional factors
which might have hampered the bioavailability of
nutrients. Agarwal and Sharma (2013) reported that
whole LS seed flour contain tannins, phytic acids,
oxalic acid and cyanogens which might obstruct the
bioavailability of nutrients. Maternal exposure to
inadequate or excessive amount of some nutrients may
cause malformations or irreversible effects, stillbirth,
early death or live birth of weak kids.
LS seed inclusion into the diet of female rabbits
increased number of runts and incidence of stillbirth.
Furthermore, the occurrence of stillbirth was bimodal
with highest incidence seen in the 5%- and 10%-LS
seed inclusion. In addition, all runt kids died within 48
hours of delivery. The death of the runt kids is
attributed to the extremely low birth weight. Low birth
weight kids are physiologically immature when
compared to littermates of average birth weights. They
are also at greater risk from hypothermia and cannot
compete well for milk against their larger litter mates.
The number of stillbirth and runt observed in rabbits
fed LS in this present study may indicate fetal
developmental toxicity effects of LS seed. This may be
attributed to the abundance of phytosterols in LS seed
and the most potent, phytoestrogens. Phytoestrogens
possess goitrogenic activity, a reported property of LS
(Ensminger et al., 1994; Okpala, 2015). A goitrogen is
a substance that disrupts the production of thyroid
hormones by interfering with the uptake of iodine in the
thyroid gland, thereby causing hypothyroidism.
Uncontrolled hypothyroidism or thyroid failure during
pregnancy can lead to low birth weight and stillbirth
(Abalovich et al., 2002; Brent, 2012). This may due to
reduced foetal thyroxine which may cause disruption in
the pituitary-thyroid gland axis of the new born. The
foetuses are highly sensitive to environmental chemical
exposures thereby affecting organogenesis, rapid
growth and extensive tissue differentiation that occur
during these developmental periods and therefore small
perturbations can have important consequences. In
addition, metabolic processing and elimination
mechanisms are immature in the foetus, so
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Lepidium sativum and pregnancy outcome in rabbits
J. Afr. Ass. Physiol. Sci. 6 (1): 2018 Imade et al.
detoxification is inefficient (Beath, 2003; Chen et al.,
2006). Therefore, a culmination of these factors may
precipitate the stillbirth observed in the LS-
supplemented rabbits in this study. Further studies
should be done on the reversible effects and the impacts
of LS seed on organ systems of offspring born/survived
by mother fed LS based diet during pregnancy in
rabbit.
CONCLUSION
The results of the current study showed that Lepidium
sativum seed inclusion in the diet is capable of
stimulating LH and increasing conception rate in
female rabbit through the secondary metabolites but
however may pose harmful effects on developing rabbit
foetus due to low birth weight, reduction in live born
kits, high rate of stillbirths observed.
ACKNOWLEDGEMENTS
St. Cloud State University (SCSU), St. Cloud,
Minnesota, USA, is hereby acknowledged for
supplying the needed laboratory materials required for
timely completion of this study. Thanks also go to Aja,
Frank, Shorinmade Abiola, Olawale Abiola and Ojo
Oluwaseun for their support which helped a lot during
this research work.
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