Mariano Illera

Steroid-level response to insulin-like growth factor-1 in oocytes matured in vitro.

The objective was to establish the influence of insulin-like growth factor-1 (IGF-1) on steroid production and nuclear maturation during oocyte in vitro maturation (IVM). Immature-selected rabbit follicular oocytes, divided as cumulus-oocyte complexes (COC) and denuded oocytes (DO), were cultured in Bracketts medium with different concentrations of IGF-1 at 0, 50, 100 and 200 ng/ml. After 8 and 16 h of culture, the oocytes were assessed for nuclear maturation by acetic-orcein stain, and media were analyzed by enzyme-immunoassay (EIA) for 17 beta-estradiol (E), progesterone (P), androstenedione (A) and testosterone (T) content. After culture treatments with IGF-1 significantly increased (P < 0.01) the incidence of nuclear activation (germinal vesicle breakdown stage, GVBD) and nuclear maturation (metaphase II stage); maximum stimulation occurred at 100 ng IGF-1/ml (86.9 vs. 49.3% in control). Compared to controls, the presence of IGF-1 in cultures was associated with a significant increase of E and A production by COCs (P < 0.01). However, P and T levels were not significantly influenced by the IGF-1. In addition, positive correlations between E/T and E/A ratios and nuclear maturation rates were only found in the IGF-1 treatments. Regarding the DOs, neither positive effects in nuclear maturation rates nor increase of steroid levels in culture were observed for any treatment. These results suggest that: (1) IGF-1 had a significant effect on E and A production during oocyte maturation; (2) the addition of IGF-1 enhanced nuclear maturation significantly in rabbit oocytes; and (3) all these effects are only possible in oocytes surrounded by cumulus cells.

The effect of clenbuterol on adrenal function in rats

The use of anabolic agents is illegal in the European Community but the effect of these agents on animal welfare is not well documented. The aim of this study was to evaluate whether the administration of anabolic agents, such as clenbuterol, causes stressful effects in rats, as reflected by the adrenal function. Anabolic doses of clenbuterol (1 mg kg-1, 99% purity) were administered orally by stomach tube daily for 45 d to female Long Evans rats (250-300 g, n = 50). Twenty-five animals were used as controls. Blood samples were collected from the jugular vein in anaesthetised animals (ketamine and xylazine). At the end of the experiment, the animals were sacrificed and the adrenal glands were removed. Hormonal levels were measured by an enzyme immunoassay previously validated for this species. Hormonal levels of cortisol and corticosterone, and histopathological analysis, were used as indicators of the adrenal function. Increased corticosterone and cortisol secretion was found in the treated group (p < 0.001), both in adrenal homogenates and peripheral blood samples, compared with control animals. Higher relative adrenal gland weight (adrenal gland-to-body weight ratio) was also found in the treated group (p < 0.01). The major histopathological finding was the presence of hyperplasia in the adrenocortical cells. It was concluded that the administration of an anabolic dose of clenbuterol causes a hyperstimulation of adrenal gland secretion that could adversely affect animal welfare.

Amplified androstenedione enzymeimmunoassay for the diagnosis of cryptorchidism in the male horse: comparison with testosterone and estrone sulphate methods

An amplified enzymeimmunoassay (EIA) was validated for androstenedione in the serum of male horses. We will use the assay as a tool for the diagnosis of equine cryptorchidism. We will compare androstenedione EIA to the currently used methods (testosterone and estrone sulphate determinations). The study was conducted on 115 horses of pure Spanish and Arabian breeds, that included 30 geldings, 60 bilateral cryptorchids and 25 stallions. Androstenedione standard curve covered a range between 0 and 1 ng per well. Low detection limit was 1.54 pg/ml. Intra- and inter-assay coefficients of variation (CV%) were <8.2 and <9.3, respectively (n=10). Recovery rate of known androstenedione concentrations averaged from 96.62+/-2.69 to 97.63+/-1.87%. Androstenedione mean+/-S.E. serum concentrations were 10.52+/-1.36 ng/ml in stallions (n=25), 0.51+/-0.04 ng/ml in cryptorchids (n=60), and 0.03+/-0.01 ng/ml in geldings (n=30). Diagnostic validation parameters in basal samples showed for estrone sulphate the lower positive predictive value (0.85) with the higher number of false positives, and lower specificity (0.84). Testosterone showed the higher number of false negatives with a negative predictive value of 0.85, and lower sensitivity (0.85). Among the three hormones evaluated, androstenedione presented the best results with the smaller number of horses diagnosed as false positives (0.93) or negatives (0.91). This technique also resulted in higher sensitivity, specificity and efficiency over the other two methods assayed. We concluded that our amplified EIA is a highly sensitive and specific assay that provides a rapid, simple, and inexpensive alternative to other methods.

Determination of follicular fluid estradiol levels by enzyme-linked immunosorbent assay

A direct competitive heterologous enzyme-linked immunosorbent assay (ELISA) was developed and validated to determine follicular fluid estradiol levels of different antral follicular sizes (small, medium, and large) obtained from the ovaries of heifers in the follicular phase of the estrous cycle. Polyclonal antiestradiol antibodies were raised in New Zealand White rabbits using 6-keto-1,3,5(10)-estratriene-3,17 beta-diol 6-carboxymethyloxime: bovine serum albumin as immunogen and characterized by the usual methods. Horseradish peroxidase conjugated to 1,3,5(10)-estratriene-3,17 beta-diol 3-hemisuccinate was used as a label. The concentration range used for the standard curve was 0 to 1 ng per well. The low detection limit of the technique was 0.3 pg per well with a sensitivity at 50% binding of 93.62 pg per well. Intraassay and interassay CV (%) were < 5.3% and < 7.0%, respectively (n = 10). The recovery rate of the known estradiol concentrations added to follicular fluid averaged 96.1 +/- 1.3%. Compared with a radioimmunoassay (RIA), the values of ELISA were highly correlated (r = 0.99, P < 0.005). This assay was used to quantify follicular fluid estradiol concentrations without previous extraction in three antral follicular sizes; small (< 5 mm), medium (5.1 to 10 mm), and large (10.1 to 20 mm). The mean +/- SE follicular fluid estradiol concentrations were 77 +/- 5.2 ng/ml (n = 490) in small follicles, 111 +/- 19 ng/ml (n = 65) in medium follicles, and 496 +/- 144 ng/ml (n = 45) in large follicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoflavones and their effects on the onset of puberty in male Wistar rats.

This study was performed to determine how two of the most important isoflavones, genistein and daidzein, affect the gonadal axis in male prepuberal rats. One hundred and seventy‐five prepuberal male Wistar rats were allocated into seven groups: one control group and six experimental groups that were orally administered a high or low dose of genistein, daidzein or a mixture of both. Testosterone determination was assayed by EIA. The testes and body weights were measured, and the histology of the epididymis with the sperm content and epididymal sperm count were evaluated. In the control group, we observed an increase in the serum testosterone levels (>2.5 ng ml−1) at the third week (52 days), which corresponded to the onset of puberty in these rats. The same increase in serum testosterone levels was observed at the fourth week in rats that received low doses of isoflavones; therefore, we concluded that the onset of puberty was delayed. At high doses, there was no significant increase in testosterone levels, which could be related to the fact that these male rats did not reach puberty. These findings were supported by the results obtained from the analysis of the epididymal content as well as the testes/body weight ratio.

The Effects of Isoflavones on Androgens and Glucocorticoids During Puberty on Male Wistar Rats

Isoflavones are the most common phytoestrogens found in human diets. However, it is still not clear whether isoflavones have effects on the reproductive and the endocrine systems under normal dietary intake and overdose. The aim of this study was to determine how the most important isoflavones, genistein and daidzein, affect androgen and glucorticoid levels on male prepuberal rats. A hundred and seventy-five 30-day-old male Wistar rats were dosed orally by stomach tube every day for 35 days, with saline solution, low and high doses of genistein, daidzein and a mixture of both. Serum samples were analysed by an enzyme immunoassay for hormone determinations. In control group, there was a peak of testosterone (T) and dihydrotestosterone levels associated to the onset of puberty, at the third week. However, in low-dose groups, the same peak was found at the fourth week (p < 0.05), indicating a delay in the onset of puberty in these groups. Moreover, high doses groups serum androgen levels were significantly lower (p < 0.05) than the control group from the first week until fifth week. This fact was supported by a epididymal histological analysis that indicate in low doses there were several content of spermatozoa at fourth week and in high doses there were few content of spermatozoa. Besides, corticosterone levels followed the same pattern of androgens in all groups. We can conclude that oral administration of isoflavones in male rats decreased the secretion of androgens and glucocorticoids causing a delay in the onset of puberty and may cause physiological and developmental problems.