L. Forró szerk.: Miscellanea Zoologica Hungarica 7. 1992 (Budapest, 1992)
Gubányi, A., Kéri, Gy. , Fehér, T.; Pekli, J.: A chicken GnRH analogue (Folligen) stimulates plasma testosterone level and sexual activity in the male marsh frog during and out of spawning season - Preliminary notes
among several environmental cues this steroid may regulate post-reproductive refractoriness through the central nervous system by a negative feedback mechanism. In recent years several new synthetic GnRHas have been developed in our laboratory and tested on frogs, fishes and mammals (Kéri et al. 1990, Gubányi et al. 1991). A chicken I. GnRH analogue [ D .Phe 6 ,Gln 8 ,desGly 10 ]-GnRH-ethylamide, named Folligen, was the most potent among them, which stimulated follicular maturation, ovulation and spermatogenesis. The scope of our investigation was to test the effects of Folligen (GnRHa) on testosterone and estradiol productions and sexual activity of Rana ridibunda in different seasons. Material and methods Animals and treatment: Adult male marsh frogs for summer and winter experiments (Rana ridibunda Pall., 65 gram±16 STD) were purchased from the Experimental Frog Farm (MITEX Kft, Lébénymiklós, Hungary) in 1989 and kept in small ponds before investigations. The frogs tested in spring were collected in the vicinity of Rétimajor during the spawning season in April, 1990. All the frogs used for experiments were fed by crickets (Gryllus bimaculatus) ad libitum. Female frogs were used for testing amplexing behaviour of male frogs in each experiment. Visually three different stages of sexual behaviour were investigated: chorusing of males, fighting between males and amplexing. Winter experiment: Twenty male frogs, tested in winter were divided into two groups (10 individuals per group) and water temperatures gradually raised to 22±1 °C on 10 light/14 dark daylight regime. The first group was administered daily with GnRHa (Folligen) in the dorsal sac at 8 am for three days (1st day: 250 ng/g BW, 2nd day: 500 ng/g BW, 3rd day: 1000 ng/g BW). The second group was treated only with saline. Blood samples were taken on the third day at 2 pm. Spring experiment: Ten male frogs, collected during the breeding season were treated daily (at 8 am) with GnRHa for three days (1st day: 0.25 ng/g BW, 2nd day: 0.5 ng/g BW, 3rd day: lng/g BW). The water temperature was between 21-22 °C. Blood samples were taken from every individual one hour before the first injection and 5 hours after the last treatment. Summer experiment: 25 male frogs, tested in summer were grouped (10 specimens per treated groups and five individuals for controls). Blood sampling was carried out as in exp. 2. Groups were injected daily with 100 ng and 1 ug GnRHa per animal for six days, respectively. Controls were injected with saline. Frogs were kept at 19±1 °C and 10L/14D light-dark cycle. Chemicals: The GnRHa, Folligen [D-Phe 6 ,Gln 8 ,desGly 10 ]-GnRH-ethylamide was synthesized in our laboratory. Blood sampling: Blood was collected from a blood vessel on a hind limb using a micropipette (Wijnands & van Gelder 1976). Samples were centrifuged (15 min, 4500 rpm). Thereafter, about 400500 u\ plasma was diluted with Ringer's solution up to one millilitre and stored at -25 °C until processing. Hormone measurement: Determination of Plasma testosterone (T) and estradiol-17 (E) were carried out by radioimmunoassay (RIA) (Bodrogi & Fehér 1980, Poteczin et al. 1984). Statistics: The significance of differences was evaluated using Student's T-test and Mann-Whitney U-test.
