Association between the Seminal Vesicle Weight and Certain Steroids in Buffaloes (bubalus Bubalis)

Introduction androgen is converted to estrogens by an enzyme aromatase cytochrome P450 (P450arom) which has a Seminal vesicles (vesicular gland) are firm and central role in organizing neuroendocrine function and lobulated structures located adjacent to the neck of regulates a variety of reproductive and social behaviours bladder and lateral to ampulla [1]. As age of the animal [6]. Mammalian semen is known to contain a big increases, the size of seminal vesicles also increases in variety of chemical elements [7] produced under the terms of its biometry, thus there is a positive correlation influence of androgens. Their influence on spermatozoa between age of animal and size of seminal vesicles [2]. viability has been extensively studied in animals as Furthermore, the effect of age and breed on the seminal well as in humans. The P450arom protein is highly vesicle's weight and its secretory activity has been conserved in its function, if not its peptide sequence [8] reported [3]. Interestingly, its secretory activity is and is expressed in the brain of all vertebrates dependent on the level of androgen in male reproductive studied, including amphibians, birds, fish, and reptiles tract. [9]. A significant correlation between the growth and In mammals, The HPG axis drives reproduction: the secretory activity of the seminal vesicles controlled Hypothalamus secretes Gonadotrophin Releasing by the level of androgens: testosterone, androstenedione Hormone (GnRH), GnRH stimulates the gonadotroph and dihydrotestosterone, has been reported [10]. cells of pituitary to secrete Follicular Stimulating Presence of high levels of estrogen has been reported in Hormone (FSH) and Luteinizing Hormone (LH), and semen compared to blood plasma, even after frequent in turn these two hormones regulate the gonadal excessive ejaculations [11], thus it suggests that function in both sexes [4]. Steroids like testosterone estrogen is being secreted into semen mostly from and estrogen are no longer considered male specific accessory glands. Currently, researchers indicate that hormones as both hormones are important in male and germ cells also synthesize estrogen, and possibly serve female. In males, testosterone produced by Leydig as the major source of estrogen in the male cells is converted to estradiol in the sertoli cells under reproductive tract [12]. Together with the existence of a the influence of aromatase [5]. Also in brain, the functional aromatase, the intracrine role of estrogens is important in production of immature germ cells as well as during ? nal steps of spermatozoa maturation [13]. It was …


Introduction
androgen is converted to estrogens by an enzyme aromatase cytochrome P450 (P450arom) which has a Seminal vesicles (vesicular gland) are firm and central role in organizing neuroendocrine function and lobulated structures located adjacent to the neck of regulates a variety of reproductive and social behaviours bladder and lateral to ampulla [1].As age of the animal [6].Mammalian semen is known to contain a big increases, the size of seminal vesicles also increases in variety of chemical elements [7] produced under the terms of its biometry, thus there is a positive correlation influence of androgens.Their influence on spermatozoa between age of animal and size of seminal vesicles [2].
viability has been extensively studied in animals as Furthermore, the effect of age and breed on the seminal well as in humans.The P450arom protein is highly vesicle's weight and its secretory activity has been conserved in its function, if not its peptide sequence [8] reported [3].Interestingly, its secretory activity is and is expressed in the brain of all vertebrates dependent on the level of androgen in male reproductive studied, including amphibians, birds, fish, and reptiles tract.
[9].A significant correlation between the growth and In mammals, The HPG axis drives reproduction: the secretory activity of the seminal vesicles controlled Hypothalamus secretes Gonadotrophin Releasing by the level of androgens: testosterone, androstenedione Hormone (GnRH), GnRH stimulates the gonadotroph and dihydrotestosterone, has been reported [10].cells of pituitary to secrete Follicular Stimulating Presence of high levels of estrogen has been reported in Hormone (FSH) and Luteinizing Hormone (LH), and semen compared to blood plasma, even after frequent in turn these two hormones regulate the gonadal excessive ejaculations [11], thus it suggests that function in both sexes [4].Steroids like testosterone estrogen is being secreted into semen mostly from and estrogen are no longer considered male specific accessory glands.Currently, researchers indicate that hormones as both hormones are important in male and germ cells also synthesize estrogen, and possibly serve female.In males, testosterone produced by Leydig as the major source of estrogen in the male cells is converted to estradiol in the sertoli cells under reproductive tract [12].Together with the existence of a the influence of aromatase [5].Also in brain, the functional aromatase, the intracrine role of estrogens is important in production of immature germ cells as well as during ?nal steps of spermatozoa maturation [13].It was also demonstrated that during fetal and neonatal life, estrogens are involved in control of gametogenesis, the seminal vesicles were cleaned and then carefully promoting germ cell and seminiferous tubule cleared the tissue debris, fatty tissue, fascia and other development and in the regulation of fetal Leydig cell part from seminal vesicles.Then organs were cleaned steroidogenesis [14].Testosterone either alone or with again (with normal saline, 0.9% NaCl, GR Grade, estradiol 17 β has been reported to maintain sperm Merck, India), soaked and dried with tissue paper motility for 14 days after castration [15].However, before processing.The gross morphological there are few studies which have shown concentration parameters of seminal vesicles (right and left separately) of steroids in seminal vesicle tissue.In buffaloes, were recorded.overall mean testosterone concentration in seminal Seminal vesicles' (right and left) weight was vesicle flushed and squeezed fluid has been reported as recorded separately before and after squeezing the 0.32±0.06ng/ml and 0.24±0.03ng/ml respectively fluid using electronic balance (A and D Company [16].In Holstein-Friesian bulls, estrogen concentration Limited, Japan).On the basis of mean weight, seminal has been reported in tissues of seminal vesicle as 0.008 vesicles were divided into three groups as group I (<5 ± 0.002 ng/gm [17].The estrogen with lower dose of 2 gm), group II (5-8 gm) and group III (>8 gm).microgram/ml showed a beneficial effect on motility Seminal vesicle fluid collection: Seminal vesicles and acrosomal integrity of bull sperm in-vitro [18].
(right and left) were squeezed and flushed (using The information regarding the testosterone potassium phosphate buffer, 50 mM, pH 7.4).concentration in seminal vesicle fluid and in tissues of buffalo bulls [16] and levels of estrogen in bulls [17] Squeezing: Each right and left seminal vesicle was has been previously reported.In this study, we have squeezed separately and obtained volume of seminal established an association between the mean seminal vesicle fluid was collected in the test tube.vesicle weight and its hormonal content as an indicator Flushing of seminal vesicles: Immediately after of fertility in buffaloes.
squeezing and weighing, each seminal vesicle was

Materials and Methods
incised longitudinally and transversely at all length and width in each seminal vesicular duct and lobule using The concentration of testosterone from buffaloes' sacrificed at local abattoir, irrespective and estrogen in supernatant fluid was determined by of age, breed and body weight.Immediately after Radioimmunoassay (RIA) kit (Immunotech, France) sacrifice (within an hour), the seminal vesicles along and radioactivity was counted by COBRA II, Auto with other tissues were collected and ligated at the base Gamma Counter, Packard, as per the standard protocol of the seminal vesicular duct to prevent fluid loss and provided with kit.finally placed in a polythene bag (with sealing device) and transferred into the thermos flask containing ice Statistical analysis: All the data were statistically cubes and then transported to laboratory.In laboratory, analyzed by SPSS 16.0 using one way ANOVA.The samples were collected from local slaughter house where animals are sacrificed with Government approval.We have not sacrificed any animal for our study so there are no ethical issues in this study.Mean with different superscripts in a row (a, b, c) and in a column (A,B) differ significantly at p<0.05 and p<0.01 respectively.On multiple comparison, the mean differ significantly between I vs II and I vs III at p<0.028 and p<0.035 respectively.Group-1: Group-3: (n=7), Group-4: (n=30) (n=9), Group-2: (n=14),

Parameters
Group [20].In our study, testosterone concentration was and Fig- 1) but the difference was significant (p<0.05)significantly (p<0.05)correlated (r=0.365) with only between group I and II.However, testosterone seminal vesicle weight.Similarly, in bovines, pituitary concentration in left seminal vesicle was significantly gland size increases from, 0.57±0.05gmat birth to (p<0.05) higher in group III compared to I and II.
1.77±0.05gmat one year of age [21].In present study, Values in group II were not significantly differing from we found higher weight of seminal vesicle with higher group I or III.The difference was significant (p<0.05)testosterone concentration which influences the only between group III and I. On the other hand, in secretory activity and thus there will be secretion of pooled and mean seminal vesicle fluid, testosterone several androgen dependent proteins serving as a useful concentration showed significantly (p<0.05)lower marker of seminal vesicle activity [22].Similarly, in value in group I compared to group II and III.The bulls, significant correlation between the growth and variation within group I and II between right and left the secretory activity of the seminal vesicles has been seminal vesicles were non-significant.However, testoreported [10].Secretions of seminal vesicles involves, sterone concentration was significantly (p<0.01)higher proteins that bind to sperm at ejaculation and modify in left seminal vesicle in group III.The intra-assay the sperm membrane by removing cholesterol and coefficient of variation was 7.11%.Lower testosterone phospholipids, which may adversely affect the ability concentration in flushed and squeezed seminal vesicle of sperm to be preserved [23].In four year old bull, fluid of buffaloes invitro has been reported [17] compared higher size of seminal vesicles has been reported [2] to our study.Testosterone is the major androgen compared to 2 and 3 year old bulls thus indicating produced from leydig cells under the influence of LH.
positive correlation between the age and size of seminal Androgens are important hormones for expression of vesicles.Similarly in the present study, increase in male phenotypes and also have characteristic role level of steroids with increase in weight of seminal during male sexual differentiation during development vesicle might be due to age of animal, as age of animal and also during initiation and maintenance of spermaincreases there is increase in size of seminal vesicles.Estrogen levels in seminal vesicle fluid: There was compared to group I (with <58 gm weight of seminal increase in estrogen concentration from group I to II vesicles).As already mentioned, the secretions of and to III, but the difference is significant (p<0.05)only seminal vesicles are important for obtaining fertilizing between group I and III in right seminal vesicle fluid, ability of spermatozoa.Thus present study showed that with overall mean value of 9.32±2.24pg/ml (Table -2 seminal vesicle secretion is dependent on level of and Fig- 2).Similar trend was also observed in left, androgens.Therefore, steroids estimation could be used pooled and mean seminal vesicle fluid estrogen as a parameter in selection of breeding bull along with concentration from group I to II to III with overall mean other factors.value of 8.18±1.65 pg/ml, 17.51±3.68pg/seminal Authors' contributions vesicle and 8.75±1.84pg/ml, respectively.On multiple SS planned and carried out research work for his MVSc comparison of overall mean seminal vesicle fluid thesis programme in collaboration with advisory estrogen concentration showed significantly (p<0.005)members and guide (SM).GS guided and provided higher concentration in group III compared to group I.
facilities in estimation of steroids using RIA.MRV Comparative evaluation of right and left seminal performed statistical analysis.All authors read and vesicle fluid estrogen concentration showed nonapproved the final manuscript.significant changes within groups, however, estrogen was non-significantly higher in right seminal vesicle in

Figure- 1 .Figure- 2 .
Figure-1.Testosterone concentration in right, left, pooled (ng/seminal vesicle) and mean seminal vesicle fluid in three seminal vesicle mean weight groups in buffalo in-vitro.Values with different letters are statistically significant (P< 0.05)

Table - 1
. Mean ± SE of testosterone concentration in three seminal vesicle weight (gm) groups in buffaloes invitro

Table - 2
. Mean ± SE of estrogen concentration in three seminal vesicle weight (gm) groups in buffaloes invitro