Effects of Kamdhenu Ark and Active Immunization by Gonadotropin
Releasing Hormone Conjugate (GnRH-BSA) on Gonadosomatic
Indices (GSI) and Sperm Parameters in Male Mus musculus
Javid Ahmad Ganaie, Varsha Gautam, Vinoy Kumar Shrivastava
*
- Endocrinology Laboratory, Department of Biosciences, Barkatullah University, Bhopal, India
Abstract
Background: Active immunization against GnRH decreases the secretion of gonadotropins and causes cessation of gonadal function, thereby, inducing infertility. Based
on the immunoenhancing activity of Kamdhenu ark (distilled cow urine), this study
was performed to evaluate its effects on the gonadosomatic indices (GSI) and sperm
parameters in male mice receiving a GnRH contraceptive vaccine.
Methods: Sixty adult male mice of Parke’s strain were divided into three groups of
twenty. Group I served as the controls, while group II was immunized by GnRH-BSA
conjugate (50/0.2/35 µg/ml/g BW) by four intraperitoneal injections at different intervals on days 1, 30, 60 and 90. However, group III was supplemented daily by oral
Kamdhenu ark (100 ppm) along with GnRH-BSA immunizations. The animals were
sacrificed after 30, 60, 90 and 120 days and their testis and epididymis were dissected
out weighed and semen analysis was performed.
Results: GSI values, sperm motility, sperm count and sperm morphology in male Mus
musculus were decreased significantly in all the experimental groups as compared to
the control group (p<0.01). Kamdhenu ark significantly enhanced the effect of GnRH
vaccine on the aforesaid parameters especially in 90 and 120 days treated groups
(p<0.05).
Conclusion: The changes witnessed in sperm parameters suggested that the GnRHBSA immunization suppressed the activities of gonadotropins and testosterone directly through hypothalamo-hypophysial-gonadal axis and indirectly by acting on the
testes which may modulate the sperm morphology, sperm count and motility. However, Kamdhenu ark seems to have enhanced these effects because of its immunemodulatory properties too.
Keywords: GnRH-BSA, Gonadosomatic indices (GSI), Immunization, Kamdhenu ark, Mus
musculus, Sperm parameters.
To cite this article: Ganaie JA, Gautam V, Shrivastava VK. Effects of Kamdhenu Ark and Active
Immunization by Gonadotropin Releasing Hormone Conjugate (GnRH-BSA) on Gonadosomatic Indices
(GSI) and Sperm Parameters in Male Mus musculus. J Reprod Infertil. 2011;12(1):3-7.
Background
onadotropin-releasing hormone (GnRH) con-
trols the production of gonadotropins, thereby having an orchestrating effect on the reproductive hormone cascade and spermatogenesis (1).
Active immunization against GnRH has successfully suppressed the secretion of gonadotropins
and decreased sperm production, follicular development, ovulation and conception in male and female mammals (2, 3). Vaccination against GnRH
blocks the hypothalamic-pituitary- gonadal axis.
Therefore, it can be used as an alternative for
castration and fertility control in farm animals,
companion animals and wildlife species (4-6).
Application of GnRH vaccination in humans has
been suggested for controlling fertility-related
endocrine disorders and gonadal steroid-dependent diseases (7). Active immunization of adult
animals against GnRH causes the loss of synthesis
and secretion of gonadotropins and cessation of
gonadal function as long as the antibody titers
* Corresponding Author:
Vinoy Kumar Shrivastava,
Endocrinology
Laboratory, Department
of Bioscience, Barkatullah
University, Bhopal (M.P.)
India.
E-mail:
vinoyks2001@yahoo.com
Received: May 9, 2010
Accepted: Aug. 10, 2010 J Reprod Infertil, Vol 12, No 1, Jan/ Mar 2011
JRI Kamdhenu ark and GnRH-BSA immunization
4
remain elevated (8).
It has been reported that cow urine contains all
beneficial elements such as chemical properties,
potentialities and constituents that are capable of
removing all the ill effects and imbalances of
body caused by various infectious agents and
toxicants. In this way, it ensures a protection
against various ailments including the most
dreaded diseases like cancer, diabetes, hepatitis
etc. (9). Kamdhenu ark (distilled cow urine) has
been reported as a strong immunomodulator and
bioenhancer by various researchers (10, 11).
Experimental studies of Rangasamy and Kaliappan revealed the protective effects of cow urine on
haematological, serum biochemical parameters
and immune status of broilers (12).
The present study attempts to evaluate the
effects of GnRH-BSA immunization on gonadosomatic indices (GSI) and sperm parameters in
male mice and to examine the modulatory role of
Kamdhenu ark following the immunization.
Methods
Sixty adult male mice, Mus musculus, of Parke’s
strain (P), weighing 30±5 g were used in the
study. The animals were divided into three groups
of twenty. The mice in Group I served as the
controls, receiving intraperitoneal Phosphate
Buffered Saline (PBS) injections (100 µl) on the
1
st
, 30
th
, 60
th
and 90
th
days, while the mice in
group II were immunized by GnRH-BSA conjugate (50/0.2/35 µg/ml/g BW) (Sigma-Aldrich,
USA) dissolved in 100 µl of phosphate buffered
solution (0.01 N) emulsified with an equal volume
(100 µl) of Freund’s adjuvant (Sigma Aldrich,
USA). GnRH-BSA injections were given intraperitoneally at different intervals, i.e. on days 1
st
,
30
th
, 60
th
and 90
th
However, the mice in group III .
were supplemented with daily Kamdhenu ark
(100 ppm) (Gaytri Shakti Peeth, India) orally
along with the intraperitoneal injections of GnRHBSA. Five animals from each group were
sacrificed in monthly intervals, i.e. on days 30, 60,
90 and 120 and their testes and epididymides were
quickly dissected. The testes were weighed for
observing gonadosomatic indices [gonad weight/
100 g BW], while the epididymides were processed for semen analysis, i.e. sperm motility,
sperm count and morphology by Prasad method
(13). Cauda epididymides were dissected out to
release sperms in normal saline (100 mg tissue/
2 ml N.S.) for sperm suspension. For studying
sperm morphology, Leishman`s stain was used
and the slides were finally observed at 400×
magnification (14).
Statistical Analysis: The collected data were analyzed through one way ANOVA and post-hoc
methods using EZANOVA software. P-values
<0.05 or <0.01 were considered significant while
values <0.001 were considered as highly significant.
Results
GSI values decreased in all the experimental
groups compared to the control group. However,
more significant decrease in GSI was observed in
the group treated by Kamdhenu ark along with
GnRH-BSA, especially in the later part of the
experiment (p<0.01) (Table 1). Moreover, sperm
motility and sperm count significantly decreased
throughout the investigation in all the treated
groups compared to the control group (p<0.01)
(Table 2). However, some mice immunized by
GnRH-BSA + Kamdhenu ark also showed decreased values for sperm motility and count than
the GnRH-BSA immunized groups (p<0.05). The
Table 1. Gonadosomatic indices (GSI) in the experimental and control groups of
male mice, Mus musculus,after different intervals
Group
GSI (gonad weight/100 g BW)
Duration
30 days 60 days 90 days 120 days
Control 0.40 ± 0.02 0.43 ± 0.05 0.50 ± 0.01 0.54 ± 0.04
GnRH-BSA 0.36 ± 0.06 0.30 ± 0.02
a
* 0.23 ± 0.00
a
0.16 ± 0.00
a
GnRH-BSA + KA 0.32±0.03 0.26± 0.01
a
0.19±0.01
ab
0.12±0.00
ab
Mean ± SEM of five animals (Accuracy of calculation up to two decimal digits)
a
= Significant difference with the controls in the same column (p< 0.01)
b
= Significant difference with GnRH-BSA groups in the same column (p< 0.01)
* = Significant differences (p< 0.05) J Reprod Infertil, Vol 12, No 1, Jan/ Mar 2011
Ganaie JA, et al. JRI
5
percentage of morphologically normal sperm decreased significantly with increased percentage of
abnormal forms of sperms, i.e. pin head, large
head, oval head, double head, head less, bent
neck, looping mid piece, coiled-tail, double-tailed,
tailless in all the experimental groups as compared
to the control group (p<0.01) (Table 3, Figure 1).
Moreover, some significant alterations in normal
sperm morphology, such as large head, headless
and pin head sperm were also observed in GnRHBSA + Kamdhenu ark treated group when compared with GnRH-BSA, especially in the later part
of the experiment (p<0.01).
Discussion
The endocrine effects of active immunization
against GnRH have been studied in a variety of
young adult male and female animals (15-17).
Experimental studies have demonstrated decreases in gonadotropins, sperm production,
follicular development, ovulation and conception
after immunization against GnRH, chemically
conjugated to a carrier protein. GnRH immuneization affected sperm motility and sperm counts
in ram lambs, boars and colts (18, 19). Several
other experimental studies have revealed the
deleterious effects of immunization against GnRH
on different sperm parameters in rats, bulls,
stallions, cats and dogs (20-24).
Cow urine has been tested for its immunomodulatory properties that enhance both cellular
and humoral immune responses (25, 26). Kamdhenu ark (distilled cow urine) has been reported to
increase the humoral immunity in rats (27).
Chauhan et al., (2004) observed that Kamdhenu
ark may modulate the immune responses because
it increases the secretion of interleukin-1 and 2
Table 2. Sperm motility and sperm count in the experimental and control groups of male mice, Mus musculus, after
different intervals
Parameters Group
Duration
30 days 60 days 90 days 120 days
Sperm Motility (%)
Control 59.00±4.33 63.76±2.77 65.05±2.31 69.60±3.19
GnRH-BSA 39.40±3.81
a
21.56±1.36
a
13.00±2.11
a
9.40±1.14
a
GnRH-BSA+ KA 33.80±1.72
a
17.06±1.00
ab
* 10.46±1.65
a
7.16±0.95
a
Sperm Count (million/ml)
Control 62.00±3.18 68.60±2.10 75.50±3.76 78.00±2.11
GnRH-BSA 49.10±2.65
a
35.22±3.21
a
21.00±2.20
a
16.42±1.78
a
GnRH-BSA + KA 44.18±2.82
a
26.12±2.00
ab
* 18.30±1.90
a
12.34±1.10
ab
*
Mean ± SEM of five animals (Accuracy of calculation up to two decimal digits)
a
= Significant difference with the controls in the same column (p< 0.01)
b
= Significant difference with the GnRH-BSA groups in the same column (p< 0.01)
* = Significant differences (p< 0.05)
Figure 1. Normal morphological sperm forms in the controls
(A) and morphologically abnormal sperms after GnRH-BSA
and Kamdhenu ark along with GnRH-BSA administration (BI) in male Mus musculus. J Reprod Infertil, Vol 12, No 1, Jan/ Mar 2011
JRI Kamdhenu ark and GnRH-BSA immunization
6
(28). Recently, Ganaie and Shrivastava reported
the modulatory effects of Kamdhenu ark on
GnRH-BSA immunization in female mice (29).
In corroboration to above studies, our study also
revealed that GnRH-BSA immunization significantly decreased the values of GSI, sperm motility, count and morphology in male Mus musculus.
The aforesaid parameters diminished more significantly in the group supplemented with Kamdhenu
ark along with GnRH-BSA immunization. All
these changes in GSI and sperm parameters
suggested that GnRH-BSA immunization could
have directly suppressed the activities of gonadotropins and testosterone through hypothalamohypophysial-gonadal axis or might have indirectly
affected the testicular tissue. However, more
significant decreases in the parameters after
Kamdhenu ark supplementation may be because
of its modulatory and bioenhancing properties.
Acknowledgement
The authors gratefully acknowledge the help and
financial support of Professor Meenakshi Benerjee, the head of Department of Biosciences,
Barkatullah University, Bhopal (M.P), India.
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Table 3. Percentage of normal and abnormal sperm morphology in the experimental and control groups of male mice, Mus musculus, after different intervals
Days Groups
Normal
(%)
Abnormal (%)
Pin head
Large
head
Oval
head
Double
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Head less Bent neck
Looping
mid piece
Coiled tail
Double
tailed
Tail-less
30
Control 58.44±2.12 5.0±0.35 1.0±0.35 4.0±0.79 1.2±0.65 4.0±1.17 3.0±0.86 4.4±1.60 1.6±0.30 0.0±0.0 2.5±1.00
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a
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60
Control 64.00±1.41 5.4±0.57 4.2±0.65 3.4±0.57 0.60±0.00 5.0±1.36 3.4±0.57 4.2±0.65 2.5±0.55 1.33±0.51 0.80±0.14
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GnRHBSA+ KA
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90
Control 67.10±1.74 4.5±0.65 2.60±1.03 2.2±0.41 0.00±0.00 3.401.71 2.60±0.90 1.60±0.83 0.00±0.00 2.60±1.03 1.0±0.21
GnRHBSA
14.24±0.48 a 13.60±1.15 5.80±0.93 6.0±1.00 1.20±0.65 10.40±0.75 12.0±0.79 17.80±1.94 2.50±1.15 1.0±0.25 8.0±0.65
GnRHBSA+ KA
12.72±0.23 ab 13.20±1.19 8.80±0.96 5.80±1.55 2.2±0.96 13.40±1.03 9.5±2.07 10.80±1.29 3.0±0.79 2.5±0.83 11.0±1.76
120
Control 72.00±1.66 2.60±0.41 2.20±0.39 1.0±0.50 0.00±0.00 2.80±1.29 1.20±0.54 2.0±0.44 1.00±0.00 0.00±0.00 2.5±1.15
GnRHBSA
10.33±0.25
a
12.80±1.55 7.00±0.79 6.00±0.79 2.80±0.96 16.00±1.36 17.20±0.96 20.60±1.95 5.00±1.11 2.80±0.41 9.50±1.68
GnRHBSA+ KA
7.95±0.40
ab
14.60±1.03 11.00±0.79 7.20±0.82 3.80±0.96 17.60±1.07 15.00±1.83 14.25±2.21 4.60±1.15 4.0±1.17 12.50±0.65
Mean ± SEM of five animals (Accuracy of calculation up to two decimal digits)
a = Significant difference with the controls in the same column (p< 0.01)
b = Significant difference with GnRH-BSA groups in the same column (p< 0.01)
* = Significant differences (p< 0.05) J Reprod Infertil, Vol 12, No 1, Jan/ Mar 2011
Ganaie JA, et al. JRI
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