CONCENTRATIONS OF CAMP, CGMP, 17 BETA-ESTRADIOL AND PROGESTERONE IN THE FOLLICULAR-FLUID OF THE LARGEST COW FOLLICLE IN THE FOLLICULAR PHASE OF SEXUAL CYCLE AND AFTER SUPEROVULATION TREATMENT WITH SERUM GONADOTROPIN

The objective of this study was to compare the concentrations of 17 beta-estradiol, progesterone, cAMP and cGMP in the follicular fluid of the largest cow follicle from the follicular phase of physiological sexual cycle and of follicles after synchronization of fut by cloprostenol (PGF2 alpha) and superovulation treatment with serum gonadotrophin (PMSG), in dependence on steroidal dominance of follicles. 2 x 25 cows, Slovak Pied x Lowland Black-Pied crossbreds with active corpus luteum, were subjected to superovulation treatment on the basis of rectal examination. Rut synchronization was achieved by cloprostenol of Czechoslovak provenience (Oestrophan Spofa), administered at the amount of 500-mu-g per dose. Serum gonadotrophin (Bioveta Concern, Ivanovice na Hane) at the amount of 2500 I. U. was administered forty-eight hours before the second dose of cloprostenol. The animals were killed in slaughter-houses 48 hours later, or 72 hours later, since administration of the second dose of cloprostenol. The phase of the sexual cycle of control animals was determined by the method after Ireland et al. (1980) on the basis of morphological appearance of corpus luteum, presence of large preovulation follicle and by means of average concentrations of progesterone in blood serum. Aspirated follicular fluid was centrifuged using a cooling centrifuge at 3000 G. After separation, the supernatant was stored in a freezer at -18-degrees-C until further treatment. 17 beta-estradiol and progesterone in the follicular fluid were determined by means of kits under the brand-names RIA-test-ESTRA (SI-125-9), or RIA-test-Prog (SI-125-6). Concentrations of cyclic nucleotids were determined by the RIA kits from the Institute vor Radioisotope Research, Production and Use (Prague), cAMP by 125J RIA kit (RIO12) and cGMP by 125J RIA (RIO42). The method after Callesen et al. (1986), based on the expression of the mutual index of progesterone and 17 beta-estradiol ratio, was used to determine the steroidal dominance of the follicles. Average concentrations of estradiol in the estrogen-dominant follicles from the physiological sexual cycle were higher (3402.2 +/- 1237.1 nmol.1(-1) compared to the concentrations in the estrogen-dominant follicles of the superovulated cows in 72 hours since the administration of the second dose of cloprostenol (1502.8 +/- 238.4 nmol.1(-1) of follicular fluid). They were, however, significantly lower than in the follicles examined 48 hourse after the administration of the second dose of cloprostenol and amounted to 12 614.0 +/- 2404.0 nmol.1(-1) of fluid, corresponding to the level of statistical significance. In the progesterone-dominant follicles higher concentrations were recorded during the follicular phase of the cycle (167.0 +/- 77.5 nmol.1(-1) of follicular fluid) compared to the follicles of animals subjected to superovulation treatment and examined 48 hours (59.13 +/- 30.0) and 72 hours (100.19 +/- 86.86 nmol.1(-1)) after the administration of the second dose of cloprostenol. Average concentrations of progesterone both in the estrogen-dominant follicles (219.9 +/- 53.5 nmol.1(-1), 883.1 +/- 355.5 nmol.-1, resp.) were lower in the follicular phase of the sexual cycle than after the superovulation treatment with PMSG, in 48 hours since the administration of the second dose of cloprostenol (490.81 +/- 125.0 nmol.1(-1), 3729.4 +/- 2857.7 nmol.1(-1), resp.), or in 72 hours (421.7 +/- 124.8 nmol.1(-1), 4436.1 +/- 489.1 nmol.1(-1), resp.) in the progesterone-dominant follicles, at the significance level P < 0.001 (Fig. 1). Concentrations of cAMP in the estrogen-dominant follicles were significantly lower 48 hours after the administration of the second dose of cloprostenol (26.17 +/- 0.4 nmol.1(-1) of follicular fluid) as well as after 72 hours (26.3 +/- 3.2 nmol.1(-1) than in the estrogen-dominant follicles and follicular phase of the sexual cycle (71.4 +/- 0.7 nmol.1(-1), in both cases at the level P < 0.01. No significant changes between the compared groups were observed for the progesterone-dominant follicles. Average concentrations of cGMP in the estrogen-dominant follicles after the superovulation treatment, 48 and 72 hours after the administration of the second dose of cloprostenol, were equal to 3.5 +/- 0.2 nmol.1(-1) and 4.8 +/- 0.03 nmol.1(-1), resp., which is in both cases significantly lower than in the same types of follicles from the follicular phase of the sexual cycle (5.9 +/- 0.03 nmol.1(-1)) at the level P < 0.01. In the progesterone-dominant follicles cGMP concentrations did not differ significantly (Fig. 2). The presented results indicate significantly lower production of cyclic nucleotids, particularly cAMP, in the follicles of the cows treated with cloprostenol and serum gonadotrophin compared to the follicles from the follicular phase of unaffected sexual cycle. Moreover, pronounced luteinization of follicles was also observed after the administration of serum gonadotrophin, which is indicated by high concentrations of progesterone both in estrogen-dominant and in progesterone dominant follicles. Marked differences in concentrations of cAMP, cGMP and P4 in follicles from the follicular phase of the sexual cycle and after the superovulation treatment with PMSG suggest the possible negative influence of serum gonadotrophin on the internal environment and development of pre-ovulation follicles and at the same time on the quality of oocytes produced by these follicles.