Assessment of prenatal ovarian and serum estrogen concentration in Nili-Ravi buffalo fetus

concentration


Introduction
Estradiol is involved in regulating follicle formation and activation in bovine fetal ovaries hence affect the size of primordial pool at birth and eventually the female reproductive potential [ [9], and bovine [10,11]. It has been reported that ovarian estradiol production varies considerably over the course of gestation; increased during early gestation when oogonia are very active mitotically, but its production decline at the beginning of the second trimester at the time of initiation of the follicles formation [1,12,13]. In bovine the biosynthesis of estradiol in the fetal ovary has been demonstrated at the time when the sex of embryonic gonads is morphologically distinguishable as early as at 45±3 days with crown to rump length of 3.0 to 3.5 cm [14, 15,16]. A causal relationship between endogenous estradiol and inhibition of follicle formation and activation has been observed in cattle [2,17]. However, no such study has been conducted in Nili-Ravi buffalo. Thus this study was designed to assess the estrogen concentration in prenatal ovary and serum of Nili-Ravi buffalo fetus. The results obtained may be used as baseline data in the assisted reproductive biotechnology to enhance the productive and reproductive performance of Nili-Ravi buffalo. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed, and validated for the hormones quantification in cow [18]. The high variation was found in the individual hormonal level of cows. May be this is due to the physiological stage of animal.

Materials and Methods Fetuses
Total of 73 fetuses of Nili-Ravi buffalo were collected for this study from Sihala Abattoir, Islamabad. After slaughtering of the animals, fetuses were collected immediately and the crown to rump (C-R) length was measured with measuring tap to assess the age of the fetus according to [19] using the formula "y = -11.27 + 0.3063 x" where y is C-R length "a" is the intercept, "b" is the regression coefficient and "x" is the age of the fetus. The fetuses available thus ranged from 51 days of age to 290 days. The measurement was approximate age of fetus was calculated according to [19], Where y is C-R length (crown rump length) "a" is the intercept "b" is the regression coefficient "x" is the age. y = -11.27 + 0.3063 x. A "v" shape incision from umbilical cord to the caudal end of the body on ventral side was given to collect the ovaries. They were studied at interval of 20 days and thus arranged in twelve groups. After collection, ovaries were stored at -20°c in deep freezer until analyzed for estrogen. Likewise, a sternal incision was given to expose the heart, posterior and anterior vena cava for collection of blood. The blood was cooled in ice, transported to the laboratory and centrifuged at 3000 rpm for 10 minutes. Serum was collected and kept at -20°C until analysis.

Estrogen assay
The radio immunoassay (RIA) for the assessment of ovarian and serum estrogen was carried out according to [20,21,22]. For ovarian tissues estrogen RIA, 15 mg of ovarian tissues were homogenized in 1 ml normal saline and the homogenates were extracted in 2.5 ml x 2 reagent grade diethyl ether and dried under air in water bath at 60°C. Ether extracts were reconstituted with 1.5 ml of steroid phosphate buffer (0.1M containing NaCl 0.9%, gelatin 0.1%, sodium aside 0.1%, PH 7.2) and 500 µl of sample was incubated with antibody 3 [H] estradiol (200 µl) for 18-24 hrs at 4°C. Following incubation 200 µl dextral coated charcoals was added to each tube. The tubes were kept for 30-35 minutes at 4°C and centrifuged at 3000 rpm for 10 minutes. Liquid scintillation counting (LSC) is the standard laboratory method to quantify the radioactivity of low energy radioisotopes, mostly beta-emitting and alpha-emitting isotopes. The sensitive LSC detection method requires specific cocktails to absorb the energy into detectable light pulses. The clear supernatant was decanted into scintillation vials and added 5 ml of scintillation fluid (0.5% perm blend 111-tris-MSB1 Packard international, Zurich Switzerland). Radioactivity was measured in Beckman LS 1801 liquid scintillation counter. All tubes were measured in duplicates. Results were calculated by WHO immunoassay data processing program. Serum was also processed for RIA in the same manner.

Statistical analysis
The results were analyzed for significance by analysis of variance test (ANOVA).

Ovarian estrogen concentration
The mean ovarian estrogen concentration assessed during fetal life from 51 to 290 days is presented in (Table 1). The overall mean estrogen concentration observed was 16.08± 1.87 pg/mg with peak value (37.49±10.8 pg/mg) at the age group of 191-210 days. Regression analysis of variance revealed a significant (P<0.05) increase in fetal ovarian estrogen concentration with advancement of fetal age in this study (Fig. 1).

Discussion
Ovarian estradiol production varies considerably over the course of gestation. It has been observed that its production increased during early gestation when oogonia are very active mitotically, but decline at the beginning of the second trimester at the time of initiation of the follicles formation and remained low till the first growing follicles appear [1, 12, 13]. Contrary to these researches, both ovarian and serum estrogen concentration significantly increased with the advancement of fetal age in Nili-Ravi buffalo. Estradiol negatively regulates follicle formation in mammals including cattle [2, 13]. The higher concentration of estrogen in fetal ovaries and serum observed in this study seems to be a cause of the relatively small size of primordial follicles reserve in buffalo [23,24]. However, in-depth investigation is needed to probe this relationship. The estrogen concentration determined in fetal serum from 146 to 290 days indicates maximum concentration on 8th month of fetal age. But in case of cow it's reported that in mixed umbilical cord blood fetal estrogen concentration is maximum on 6th month of fetal age. However these levels are much higher than reported in buffalo heifers from birth and puberty on 27-30 months of age. No information is available regarding the prenatal ovarian cholesterol in Nili-Ravi buffalo. The cholesterol concentration are low during the early fetal period observed which increase significantly (P≤0.001) with increase in fetal life reaching maximum on 271-290 days. Limited information is available regarding the fetal serum cholesterol. In Nili-Ravi buffalo fetus the mean serum cholesterol is 71.57 mg/100 ml from 130-290 days of intrauterine life. The highest values are 108.10 mg/100 ml and 99.96 mg/100 ml which are obtained during 5 to 6 month of fetal life. The serum cholesterol from the 6th month onward shows a gradual decrease and the minimum value (42.26 mg/100 ml) is observed at 290 days of fetal life. No information is available on estrogen production by buffalo fetal ovary, however, the bovine fetal ovary is steroidogenically active in vivo have been reported by someone, and this study also indicates that the buffalo ovary is active in producing steroid hormone during pregnancy as is indicated by the estrogen hormone concentration determined during various gestational periods. During the early fetal age (51-70) days ovarian estrogen concentration is low, which increase significantly (P≤0.05) with advance in fetal age reaching maximum concentration on 210 days (7 month) of fetal life.

Conclusion
It can be concluded from this study that fetal ovarian and serum estrogen concentration significantly increased with the advancing fetal age, a phenomenon contrary to other mammals.