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It was investigated on the posibility of the measurement by means of the heamagglutination inhibition reaction (HAIR) of the PMSG in horse serum and the application of early diagnosis for pregnancy with HAIR. The results were summarized as follows.
1) The PMSG protein and non-specific substances in horse serum are fractionated and separeted by neutralization with meta phospholic acid-alkali. The activity of the PMSG was roughly recovered quantitatively with this method.
2) The antigen-antibody reactions was observed among each antigen to the PMSG and between the PMSG and anti-PMSG, anti-ES by the cross-HAR. This seems to be indicated that the protein from host origin coexist as impure protein into protein substance of hormone.
The anti-ES factor was eliminated from antigen-antibody systems between the PMSG and the anti-PMSG by reaction with male horse serum lyophilized. In cross HAIR and the neutralization tests on the hormone activity, it showed specifically the positive reactions between the PMSG and anti-PMSG, while the reactions with the HCG, ES, HS and NRS were not observed.
3) It is found that the comparisons between the HAIR and the biological response shows the agreed results.
4) It was investigated on the development and persistence of the PMSG in serum with the lapse in pregnancy of horse. The time that the PMSG is demonstration in serum differs from individuals, and it can not obtained the PMSG in serum within 30 days after the fertilization. But after that, the PMSG value increased gradually and indicated the largest titer at 6090 days, but it showed the decrease with progress pregnant periods, and the PMSG titer in each horses was not obtained since 140 days after fertilization. The differences among individuals, however, was large considerably.
5) On the posibility of early diagnosis for pregnancy using the PMSG in horse serum by means of the HAIR, the applied investigation in field are performed in order to compare with biological response.
From these results, all horse serum that the biological response was negative were also negative in the HAIR. And the positive in both biological response and the HAIR indicated the high and agreed rate in 92.4%, but 7.6% of which showed susceptible positive or negative.
It seemed to be necessary to examine individual horses on these causes.

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Investigation was made on a method of bioassay for the PMSG by using immature female dd mice of 21 days.
The response of reproductive organs injected with 10.0 International Unit of PMSG gave significant differences in each time at autopsy.
Responses of uterine weight, hemorrhagic follicles and corpora lutea forming reactions after once injection subcutaneously with 10.0 International Unit of PMSG were increased at 72 and 96 hours, ovarian and uterine weight responses were great decreased at 120 hours after injection, and it was comfirmed that biological life of PMSG in dd mice was about 120 hours.
It was recognized that the responses between uterine weight, hemorrhagic follicles and corpora lutea forming reactions had higher significances and uniformity in 96 hours than in 72 hours at autopsy.

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The fertilizable life of rabbit ova following induced superovulation were examined by the methods of delayed mating. A total of 29 mature does of Japanese White breed were used in this experiment. They were caged individually for one month before use and their body weight ranged from 2.53.3kg. The induction of superovulation was made by the following method; all does were treated with subcu-taneous injection of 200IU PMS for 5 days and intramuscura injection of 0.1mg estradiol at the last PMS, followed 24 hrs. later by intravenous injection of 20 Rab. U HCG. Then at exact intervals, varing from 8 to 15 hrs. after HCG, each doe was mated with fertil male. Autopsy was performed at 36 to 40 hrs. after mating, and the ova were recovered from the fallopian tubes.
The results are summarized in Table 1 and depicted graphically in Fig. 1. The mean number of ovulation points was 34.4, range 586. A total of 828 eggs, equivalent to 83.0% of the ovulations, was recovered. The percentage of does with fertilized ova were 100% when mating took place 8 to 12 hrs. after HCG injection. However, after 12 hrs., this propotion decreased gradually until 60% at 15 hrs. The proportion of ova fertilized when mating at 8, 10, 11, 12, 13, 14 and 15hrs. after HCG injection were 86.0, 91.7, 82.4, 74.3, 25.2, 23.0 and 15.8%, respectively. The proportion of ova fertilized decreased from a maximum of 91.7% at 10hrs. to 15.8% at 15 hrs., the decrease being most pronounced after 12 hrs.

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This report has extended to the measurement of ¹⁴C-(U)-L-Leucine incorporation in rat's eggs.
The experimental procedure was as follows. Rat eggs were obtained from random-bred Wistar Strain rats. 1-cell eggs (unfertilized) were collected from immature female rat which had been treated with PMS and HCG, according to the method reported by Zarrow and Everett.
Early Blastocyst were collected by flushing of uterus at 102 hr post coitum from adult female rats.
The basic medium used in the study was Ca-free Krebs-Ringer Phoshate Buffer (pH 7.4). The same solution was used for all manipulations. The eggs were examined by microscope and the normal egg were transfered to test tube contained 0.3ml of the solution. After preincubation for 10 minutes, 0.1ml of ¹⁴C-L-Leucine was added to test tube and incubated for 0, 0.5, 1, 2 and 3 hrs.
The reaction was stopped by addition of cold trichloracetic acid (TCA) to a final concentration of 5 per cent. The acid-insoluble was washed with 5 per cent TCA on the millipore filter (SCWP 8μ), and the filter was dried completely under an infra-red lamp.
The filter were transfered to the vials for determination of radioactivity by liquid scintillation counting (Parkard Tri-Carb), using 1, 000ml toluene containing 4g 2-5-diphenyle oxazole (PPO) and 300 mg 1-4 bis 2 (4-methyl-5-phenyloxazolyl) -benzyene (dimethyl POPOP) as scintillator.
The results are summarized as follows:
1. Concentration of ¹⁴C-L-Leucine
Relationship between concentration and incorporation of ¹⁴C-L-Leucine in early blastocyst of rat were shown in Fig. 1. It is possible to count when the concentration of ¹⁴C-L-Leucine is 0.252.5μc.
2. Number of eggs
The relation of the rate of incorporation to number of eggs were shown in Fig. 2. It is possible to count when the number of eggs are from 25 to 75 in incubation with 0.5μc (Spec. ac. 0.396 mci/mM) ¹⁴C-L-Leucine.
3. Incubation time
Incorporation of ¹⁴C-L-Leucine into 1-cell stage (unfertilized) of rat egg are shown in Table 1 and Fig. 3. 1-cell (unfertilized egg) were much incorporated at 1 hr. incubation with 0.5μc ¹⁴C-L-Leucine at 37.5°C.

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[in Japanese]

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