Gonadotropin release in periovulatory heifers after GnRH analogs measured by two types of immunoassays

Abstract

This study on heifers (n = 27) compared the effects of a GnRH antagonist (Antarelix) and those of an agonist (Triptorelin) on gonadotropin release during the periovulatory period of the oestrous cycle. In three experiments (EXP I-III), an initial injection of GnRH analogs was given 48 h after a single PGF 2alpha pretreatment during the luteal phase, with a further five at 12 h intervals. A challenge by a GnRH agonist (Gonavet) was performed six hours after the last application of analogs. In EXP I (n = 9), heifers received six times 1.5 mg of Antarelix, 0.5 mg of Triptorelin, and mannitol (5%; control), respectively. In EXP II (n = 12), identical Antarelix and Triptorelin treatments were followed by a single injection of estradiol-17beta valerate (6 h after Gonavet). The dosage of Antarelix was increased to 5 mg for each injection in EXP III (n = 6). Measurement of LH in blood plasma frequently sampled was performed parallely by a competitive RIA method (EXP I + II) and by a sandwich-type electro-chemiluminescence-immunoassay (ECLIA) in EXP III. This non-isotopic technique was also used to additionally analyse FSH levels. Results of EXP I showed that the GnRH antagonist equally suppressed LH surges and ovulation. On the contrary, prior to suppression of LH levels due to down-regulation of pituitary GnRH receptors the agonist Triptorelin induced an initial increase in LH concentration which was followed by ovulation. In the control animals we observed endogenous LH surges as well as smaller elevations after the agonist (Gonavet) challenge. An increase was also observed in antagonist, but not in Triptorelin treated heifers. Pituitary GnRH receptors were not detectable in animals previously treated by the analogs, whereas concentrations between 2.2-21.0 fmol/mg protein were measured in controls. Results of EXP II confirmed the described effects of GnRH analogs. Additionally, it was shown that exogenous estradiol is able to release LH from the pituitary, although a previous treatment by a GnRH agonist had dropped the pulsatile gonadotropin secretion. Contrary to the LH pattern and despite elevated amounts of the antagonist, the mean concentration and pulse number of FSH were not influenced by the antagonist treatment (EXP III). These data confirmed that (a) the reversibly blocked pituitary function induced by a potent GnRH antagonist may be a useful tool to study gonadotropin-dependent final follicular growth as well as ovulation in cyclic heifers and (b) the novel non-isotopic ECLIA methods for the determination of FSH and LH provided practical alternatives to other immunoassay types.

Polar phospholipids from bovine endogenously oxidized low density lipoprotein interfere with follicular thecal function

“…Evidence for a comparable response among Holstein cows (with a similar age and weight) was obtained by a parallel experiment (Schneider et al 2002), indicating a circulatory LH concentration of 60 15 ng/ml (n=6) which peaked 2 h post GnRH with a decline to about 45% 2 h after the peak and then to baseline values of 0·2-9·9 ng/ml. All animals developed oestrus (=day 0 of the oestrous cycle) and were slaughtered in the abattoir of the institute 18-19 h subsequent to the depherelin treatment.…”

“…Intra-and interassay coefficients of variation were 10% and 15% respectively. The concentration of 17 -oestradiol in follicle fluids was determined by a 3 H-RIA after extraction of 17 -oestradiol by ethylether using purified rabbit antibody (Schneider et al 2002). The sensitivity was 5 pg/ml, intra-and interassay coefficients of variation were 7% and 12% respectively.…”

“…The sensitivity was 5 pg/ml, intra-and interassay coefficients of variation were 7% and 12% respectively. LH was determined in a parallel study (Schneider et al 2002) to assess the response of Holstein heifers to the GnRH analogue used to evoke the LH surge for inducing the development of the large follicles studied here. LH determination was performed by an electrochemiluminescence immunoassay (ECLIA).…”

Corpus luteum development and its morphology after aspiration of a preovulatory follicle is related to size and steroid content of the follicle in dairy cows

“…Plasma, taken on day 14 after follicle aspiration, was prepared from 6mM K-EDTA blood samples from the coccygeal vessels using centrifugation (at 4 °C, 4000 × g, 10 min) and stored (-80 °C) for analysis. Progesterone concentrations in the plasma as well as progesterone and oestradiol concentrations in the follicle fluid were measured as described previously (Schneider et al 2002). Briefly, steroid levels were estimated using a direct single purified rabbit antibody H 3 RIA, after ethylether extraction.…”

Measurement of redox potential and steroid concentrations in the follicular fluid of growing and regressing follicles of mares

“…As a modification of the original method, antisera were further purified by affinity chromatography (Schneider et al, 2002). For the determination of plasma progesterone, 50-µl aliquots were directly used in duplicates.…”