gonadotropin - releasing hormone ( gnrh ) controls the production of gonadotropins , there - by 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 ) .
therefore , it can be used as an alternative for castration and fertility control in farm animals , companion animals and wildlife species ( 46 ) .
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 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 .
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 gonado - somatic indices ( gsi ) and sperm parameters in male mice and to examine the modulatory role of kamdhenu ark following the immunization .
sixty adult male mice , mus musculus , of parke 's strain ( p ) , weighing 305 g were used in the study .
the mice in group i served as the controls , receiving intraperitoneal phosphate buffered saline ( pbs ) injections ( 100 l ) on the 1 , 30 , 60 and 90 days , while the mice in group ii were immunized by gnrh - bsa conjugate ( 50/0.2/35 g / ml / g bw ) ( sigma - aldrich , usa ) dissolved in 100 l of phosphate buffered solution ( 0.01 n ) emulsified with an equal volume ( 100 l ) of freund 's adjuvant ( sigma aldrich , usa ) .
gnrh - bsa injections were given intra - peritoneally at different intervals , i.e. on days 1 , 30 , 60 and 90 .
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 gnrh - bsa .
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 ,
leishmans stain was used and the slides were finally observed at 400 magnification ( 14 ) .
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 .
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 .
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 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 gnrh - bsa + kamdhenu ark treated group when compared with gnrh - bsa , especially in the later part of the experiment ( p<0.01 ) . 
 
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 .
gonadosomatic indices ( gsi ) in the experimental and control groups of male mice , mus musculus , after different intervals mean  sem of five animals ( accuracy of calculation up to two decimal digits ) significant difference with the controls in the same column ( p < 0.01 ) significant difference with gnrh - bsa groups in the same column ( p < 0.01 ) significant differences ( p < 0.05 ) sperm motility and sperm count in the experimental and control groups of male mice , mus musculus , after different intervals mean  sem of five animals ( accuracy of calculation up to two decimal digits ) significant difference with the controls in the same column ( p<0.01 ) significant difference with the gnrh - bsa groups in the same column ( p<0.01 ) significant differences ( p<0.05 ) percentage of normal and abnormal sperm morphology in the experimental and control groups of male mice , mus musculus , after different intervals mean  sem of five animals ( accuracy of calculation up to two decimal digits ) significant difference with the controls in the same column ( p < 0.01 ) significant difference with gnrh - bsa groups in the same column ( p < 0.01 ) significant differences ( p < 0.05 )
the endocrine effects of active immunization against gnrh have been studied in a variety of young adult male and female animals ( 1517 ) .
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 ( 2024 ) .
cow urine has been tested for its immuno - modulatory 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 ( 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 hypothalamo - hypophysial - 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 .
 backgroundactive 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.methodssixty 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 g / 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.resultsgsi 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).conclusionthe changes witnessed in sperm parameters suggested that the gnrh - bsa 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 immune - modulatory properties too . 
Background
Methods
Statistical Analysis
Results
Discussion