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Elucidation of Estradiol and Progesterone Hormonal Profiles in Goat Ovarian Cyst

Sadeesh E. M. Jinu Marry John Meena Kataria
Vol 2(1), 192-196
DOI-

Ovarian cysts are an economic problem in the goat. The primary physiological defect leading to the formation of ovarian follicular cysts in cow is a failure of the hypothalamic gonadotrophin releasing hormone (GnRH) surge to trigger the preovulatory surge of luteinizing hormone (LH) in response to oestradiol although there is no published data regarding the cause of ovrian cyst in goat.. The objective of the present study was to determine the estradiol and progesterone concentration in cystic follicular fluid of goat. The enzyme immunoassay procedure for progesterone and estradiol was performed using estradiol ELISA kit and progesterone ELISA kit respectively. Hormonal data showed higher concentration of estradiol in large follicles (1.764±0.26) as compared to cystic follicles (0.968±0.063) and Progesterone concentrations however were highest in large follicle(1.374±.0773) followed by cystic follicles (5.754±0.631).


Keywords : Goat Ovarian cysts ELISA Estradiol Progesterone

Introduction

Ovarian cysts are one of the important causes of infertility in goats (Kutty et al., 1997) and the incidence of caprine ovarian cyst in an abattoir survey was reported to be 12% (Moreira et al., 1991). Ovarian cysts are an economic problem in the goat especially because goats are seasonal breeders. Affected goats without treatment will be barren until the next breeding season. The primary physiological defect leading to the formation of ovarian follicular cysts in cow is a failure of the hypothalamic gonadotrophin releasing hormone (GnRH) surge to trigger the preovulatory surge of luteinizing hormone (LH) in response to oestradiol (Silvia et al., 2002). Since limited information is available regarding the cystic follicle in small ruminant species, the primary cause of cysts is poorly understood in these species. Therefore, the objective of this study was to determine the estradiol and progesterone concentration in cystic follicular fluid of goat.

Materials and Methods

Goat cystic and normal ovaries were obtained from commercial abattoir, Delhi. Within 10-20 min after slaughter, ovaries were placed in chilled normal saline and transported to the laboratory as soon as possible. Ovaries were washed three times in saline (0.9% NaCl), immersed in 70% ethanol for 30 sec, and washed again with saline. The stromal tissues surrounding the ovaries were clipped off using scissors and ovaries were stored in saline at 4oC till further processing. Follicular fluids were isolated from cystic follicles (>12mm), and large follicles( ≥ 5 mm) , by aspiration of healthy, large follicles were assessed by the presence of vascularzed theca externa and clear amber follicular fluid with no debris and in cystic follicles granulosa layers were already exfoliated and the theca internals were thinner than those of healthy follicles(Table 1). Before aspiration, ovaries were washed at least thrice with normal saline. The follicular fluids were aspirated from cystic follicles and large antral follicles, using 18-gauge needle and sterile, non-toxic, non-pyrogenic monoejected brand syringes (Dispovan, 5.0 ml). The follicular fluid collected in   10ml- tube separately from cystic follicle and large follicles was centrifuged at a low speed (2000 rpm 15 min) to pellet the cells. Fluid part was taken out for estradiol and progesterone estimation.

Table 1: Follicular size and volume of fluid collected from normal and cystic follicle

SL.No. Type of ovarian tissues No. of Goats Diameter of follicle(mm)
1. Cystic follicles   (CF) 25 12.44±0.43
2. Large follicles (LF) 20 7.32± 0.35

 

The enzyme immunoassay procedure for progesterone and estradiol was performed using progesterone ELISA and estradiol ELISA kit respectively. Dispensed 25µl of each standard, control and samples with new dispensable tips into appropriate wells and added 200µl of enzyme conjugate to each well. Thoroughly mixed for 10 seconds and then incubated for 120 minutes at room temperature without covering the plate, after this briskly shook out the content of the wells and rinsed the wells 3 times with diluted wash solutions (400µl/well). Striked the wells sharply on the absorbent paper to remove residual droplets and added 100 µL of substrate solution to each well then incubated for 15 min at room temperature and stopped the enzymatic reaction by adding 50µl of stop solution to each well and took the optical density at 450nm with a micro titer plate reader within 10 minutes after addition of stop solution. Result were statistically analysed using single factor ANOVA

Results and Discussion

Follicular fluid was isolated from follicular cyst, luteal cyst and large follicle individually. Estradiol and progesterone concentrations were estimated in each follicle by competitive ELISA. Results showed higher concentration of estradiol in large follicles as compared to follicular cyst and luteal cyst. Progesterone concentrations however were highest in luteal cyst followed by large follicle and follicular cyst. (Table 2)

Table 2: Statistical analysis of estradiol and progesterone, present in large follicle, follicular cyst and luteal cyst

Values are expressed as mean ± SEM. Means within the column lacking a common superscripts differ significantly (P<0.01).

Follicle types Concentration (ng/ml)of Estradiol-17β/Progesterone ratio**
Estradiol-17β** Progesterone**
Large follicle 1.764±0.26a 1.374±.0773a 0.179±0.056a
Cystic follicles 0 .968±0.063b 0.51±0.037b 0.394±0.029b

 

Ovarian cyst incidence evaluations are usually performed on goat culled for reproductive Problems. Based on the observations made in slaughterhouses, results are transferred to the herd. In these assessments, the incidence of cysts would be expected to be greater as compared with females that remain in breeding herds and that are not sent to the slaughterhouse. The sampling of tissues obtained from slaughterhouses is directed at low reproductive performance animals; therefore, not being a reliable reflection of what is happening in the herd. This situation should be taken into account when comparisons with data gathered after slaughters are made. The results indicates that follicular fluid from cystic follicles of goat contained lower estradiol compared to large follicles, is consistent with that of Isobe finding (2007) in cows. On the other hand, the low progesterone levels in cystic folliles to large follicle are consistent with the finding of Halter et al. (2003) in cows. The marked changes in steroid levels in cystic follicles compared to large follicles might be due to the alteration in granulosa and thecal layers of cystic follicles compared to large healthy follicles as seen in cows (Sharaway et al., 1998). Granulosa and theca interna cells proliferate in the healthy growing follicle, whereas, the frequencies of cell proliferation in the granulosa and theca interna layers were much lower in cystic follicle (Isobe et al., 2000). Higher estradiol concentration in large follicle compared to cystic follies may be due to higher number of granulosa cells in large follicle as seen in cows (Garret et al., 1996). Since estradiol originates in granulosa cells, low levels of the granulosa cells in cyst result in a reduction in the estradiol-17β concentration in its follicular fluid. In normal endocrine condition when follicle reaches a particular size it releases an estradiol surge for the release of GnRH from hypothalamus, which is responsible for the release of LH from pituitary and this LH, is responsible for ovulation from follicle. For this GnRH and subsequent LH release need a minimum concentration of estradiol surge. Hormone concentration data from large follicles showed that they contained average 1.76 ng/ml of estradiol, but in the case of cystic follicles, it was significantly less than this, which indicated that due to deficiency of sufficient release of estradiol from cystic condition they cannot initiate sufficient response on hypothalamus for the release of GnRH and subsequently LH from pituitary for the ovulation which leads to cyst formation.

Conclusion

Deficiency of positive feedback of estrogen may be due to the lack of sufficient release of estradiol from follicle which may be a primary cause of disorder of ovulation. The present investigation indicates higher concentration of estrasdiol in large follicles as compared to cystic follicles .Further study should be done in order to find out the reason behind the low level of estradiol in cystic follicle as compared to large follicle at the genomic level by incorporating the gene for enzyme which required for estradiol formation from androstenidione within granulose cells in ovary.

References

Garrett, W.M. and Guthrie, H.D., 1996.Expression of androgen receptors and steriodogenic enzymes in relation to follicular growth and atresia following ovulation in pigs. Biol.Reprod. 55: 949-955.

Halter, T.B., Hayes S.H., Laranja, L.F., Silvia, W.J., 2003.Relationship between endogenous progesterone and follicular dynamics in lactating dairy cows with ovarian follicular cysts. Biol Reprod. 69:213-218.

Isobe N, 2007. Follicular cyst in dairy cows. J Animal Sci. 78: 1-6.

Isobe, N., Yoshimura, Y., 2000. Localization of apoptotic cells in the cystic ovarian follicles in cows. Theriogenology 54: 1159 – 1169.

Isobe, N., Kitabayashi, M., Yoshimura, Y., 2005. Micro vascular distribution and vascular endothelial growth factor expression in bovine cystic follicles.Domest. Anim Endo 29: 643 – 645

Lopez-Diaz, M.C., Bosu, W.T.K., 1992. A review of cystic ovarian degeneration in ruminants. Theriogenology. 37:1163-1168.

Moreira, E.LT.et al. (1991). Arguivos da Escola de Medicina Veterinaria da Universidade Federal de Bhia; 14:25–39.

Sharway, S.M., El-Sadaway, H.A.,1998. Levels of estrogen, progesterone, cholesterol, phospholipids and triglycerides in ovarian follicular fluid and follicular cystic fluids in buffaloes. J Egypt Vet Med Ass. 48: 671-683

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