In our ealier report, it was demonstrated that the antihormone against human chorionic gonadotrophin (Anti-HCG) might be produced easily in cattle. The present studies were designed to investigate the formation of the antihormone against pregnant mare serum gonadotrophin (Anti-PMS) in cattle receiving this hormone. Results obtained were summarized as follows:
1) Responses occurred in immature mice to PMS used in present studies.
Gonadotrophic potencies of PMS used in present study were assayed biologically by employing immature female mice and the results obtained were shown in Fig. 1. Each samples of PMS solutions at the concentrations of 1.5 IU, 3.0 IU, 6.0 IU and 12.0 IU/ml. were injected subcutaneously into inguinal region of immature female mice twice daily for 2 days (total of 1 ml.), and these animals were killed 100 hours after the first injection. Cornification of vaginal epithelial cells, formation of corpora lutea and haemorrhagic follicles in the ovaries and ovarian weight were examined, and the relations between the dosage (IU) and the responses were shown in the table presented under Fig. 1.
2) Method to determine bovine serum Anti-PMS.
Determination of bovine serum Ante-PMS was performed biologically by using immature female mice. Two parts (2/3 ml.) of undiluted bovine serum sample and 1 part (1/3 ml.) of saline solution of PMS with graded concentrations were incubated for 1 hour at 37°C, and then these mixed samples were assayed by the same method as that above mentioned for examining the potencies of PMS. This method (original method) was compared with two other methods (serumdilution method and simultaneous injection method), and no appreciable difference in PMS inhibiting titers between the original method and the two other methods could be found (Table 3 and 4).
3) Formation of Anti-PMS in cattle following injection of PMS.
Four bull calves were injected with large quantities of PMS intravenously or intramuscutlarly. One of them (No. 1) was received single injection of 180, 000 IU of PMS, and the other 3 animals (No. 2-4) were received 13-15 injections of 1, 000 IU or 2, 000 IU of PMS (total of 1, 5000-30, 000 IU) at 3 or 7 days interval. The results of the determination of Anti-PMS in the serum samples collected from these animals were shown in Table 1. In 3 animals (Nos. 1, 2 and 3) the determinations of Anti-PMS were negative on the serum samples collected at 5 to 50 days after the PMS injection. But in the remaining one (No. 4) which was injected intramuscularly with 2, 000 IU of PMS weekly for 13 times, Anti-PMS was determined to be positive on the serum sample collected an 14 days after the last injection, the inhibiting titer of the serum being 18.0 IU per ml. or more.
4) Formation of Anti-PMS in cattle following reinjection of PMS.
Four animals same as those employed in the previous experiment were reinjected with PMS 92-152 days later. Two of them were reinjected daily with 2, 000 IU of PMS for 7 days and the remaining 2 were received weekly 10 reinjections of 2, 000 IU. By these reinjections, serum Anti-PMS become positive, in all of them, their inhibiting titers being about 4.5-9.0 IU per ml. as shown in Table 2. The potencies of the serum Anti-PMS increased gradually from 5 to 10 days after the last PMS injection, reached the maximum level about 10 days later, and thereafter fell rapidly.
5) From these results, it seemed that the Anti-PMS might not be produced in cattle easily by injecting the dosage of PMS which was used generally for treating ovarian disorders.
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