Investigation on factor affecting in vitro gonadotrophin secretion from buffalo adenohypophysis

Abstract

This project was planned to investigate effects of estrogen and progesterone along with insulin in optimizing the in vitro production of pituitary gonadotropins from buffalo adenohypophysis. Attempts were also made to monitor bioactivity of in vitro produced FSH and LH in prepubertal female rabbits and buffaloes. In the first experiment, gonadotropins i.e. FSH and LH were produced by treating in vitro culture of buffalo adenohypophysis with two levels of estrogen (0.5, 1.0 mg) and progesterone (2.5, 5.0 mg). Insulin and liver extract were added in the culture medium as growth promoting factors. The amounts of FSH and LH produced were estimated by ELISA and HPLC techniques. Results showed significant (P<0.05) increase in the secretion of FSH and LH in cultures treated with estrogen and progesterone, respectively compared with control. The FSH and LH obtained were estimated to be 33.0 IU/ml and 68.67 IU/ml, respectively, in proportionate to standard of FSH being 100.0 IU/ml and LH being 1500.0 IU/ml. Experiment was repeated and sufficient amount of FSH and LH was obtained. Repeated procedure proved that the technique can be used for further production of FSH and LH. In the second experiment four groups of pre-pubertal female rabbits were treated with in vitro produced FSH≈4.0, 40.0 IU; LH≈8.5 and 85 IU (s/c), twice daily in divided dose at an interval of 12 hours for five days. Similarly, one group of rabbits was treated with placebo as control-1 and one was untreated kept as control-2. There was no treatment-related mortality; no hypersensitivity or toxic sign was seen in any of the experimental rabbit. None, of the experimental rabbits showed change in general behavior, aggressiveness/lethargy and weight. The in vitro produced FSH≈4.0, 40 IU, LH≈8.5 and 85 IU did not affect body weight and most of the blood parameters i.e. RBC and WBC count, PCV and hemoglobin (Hb) concentration of female rabbits. FSH≈4.0 and 40 IU resulted (P<0.05) on increased number of GF’s along with increased mean length, width, weight and volume of ovaries compared with rabbits treated with LH≈8.5, 85.0 IU and control. Moreover, treating rabbits with in vitro produced FSH≈40 IU, LH≈85.0 IU resulted in significantly (P<0.001) increased serum FSH (260-470 Vs 4-6 mlU/ml) and serum LH (28-48 Vs 4-5 mlU/ml), compared with rabbits of control group. In third experiment, two groups A and B (three in each group) of healthy but acyclic dairy buffaloes were treated with in vitro produced FSH≈500 IU and Folltropin-V≈5mg (s/c), twice daily and buffaloes in group C were treated with placebo/control. Treating buffaloes with in vitro produced FSH and Folltropin-V resulted in significantly (P<0.01) higher serum FSH, LH and estrogen concentration at day 5 to 6, whereas progesterone concentration was decreased (P<0.01). Day to day increase in serum FSH was also significant (P<0.01). Increase in serum LH was observed at day 5 to 6. Though the size of ovaries was increased showing numerous small GF’s, however, no one attained ovulatory capability. Hormones obtained from the culture materials showed bioactivity in the experimental animals without any toxicity.