In a recent report (1) the assay method by hemorrhagic follicles and corpora lutea forming reaction was mainly investigated.
The present paper describes the experimental results of uterine and ovarian weight response of dd mice on chorionic gonadotropin.
Following results were obtained.
1) Rank correlation was given between fresh and fixed organ weights by means of the increase in uterine and ovarian weight response of immature female dd mice to HCG. Rank correlation coefficients were 0.894 in ovaries and 0.954 in uteri, and there was a great significance at the 0.1% level of probability. It suggests that weighing procedure of fixing organs takes advantages at autopsy when there are too many fresh organs to weigh in a limitted time.
2) At the dose of 1.0IU a peak of the dose-response curve in both fresh and fixed ovarian weight was observed. A decline in weight at 1.5 IU and then again a moderate increase covering the dose level from 1.5U to 4.5IU were demonstrated. The mean ovarian weight was not more than 5mg even when an increased dose of more than 4.5 IU was administered, however. These results elucidated the two-phase response curve of the increase in ovarian weight of dd mice at a border dose level of 1.5 IU. The fact that no parallel relationship given between the dose levels of HCG used and its effect on the increase in ovarian weight using dd mice indicates unsuitability of bioassy.
3) On the otherhand, a dose-response line with a gradual increase trend in uterine weight of dd mice was observed with increasing HCG doses from 0.5IU to 6.0IU. Test of significance on the mean uterine weight for different hormone doses exclusive of 1.0 : 1.5 and 4.5 : 6.0IU showed that there was a significant difference. The regression line covered the dose range from 0.5IU to 6.0IU of HCG, and high significant evidence of regression coefficients was shown at 1-0.1% level of probability.
4) Reinvestigation was performed on hemorrhagic follicles and corpora lutea forming reaction so as to compare the result with that in previous report (1). Positive rate in hemorrhagic follicles and corpora lutea forming reaction seemed to be increasing in parallel with dose levels of HCG given. The HCG level of 4.5IU gave almost the peak of the dose-response curve, and the flat line covered the dose range of 6.0IU and 8.0IU. Considerably inactive potency was observed at the dose level below 1.0IU and the dose-response curve with an increasing trend covered the dose range of 1.0-4.5IU. These results show similar trend to those in a previous report (1) on hemorrhagic follicles and corpora lutea forming reaction.
5) No significant difference was given in uterine weight reaction and hemorrhagic follicles and corpora lutea forming reaction in repeated expriments. It seeme therefore that reactions using dd mice have reproducibility in assay of HCG.

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Probit analysis elucidated the possibility of constructing linear dose-response line from the values in hemorrhagic follicles and corpora lutea forming reaction of dd mice to HCG both in winter and in summer seasons.
By parallel line assay non-paralellism among individual dose-response lines was denied, however, . the null hypothesis that the regression coefficients are equal was not denied. Probit regression lines in winter was low on the whole in the comparison of that in summer.

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Fertility trials using pellet frozen stallion semen preserved in liquid nitrogen were conducted in 1966.
Semen, collected from 9 stallions (Percheron 3, Breton 4, Pony 2) with gel portion removed, was extended 1:1 with yolk (5 parts) -5% glucose (95 parts) solution and then centrifuged at 1, 300g, 20 min. for concentrate of semen.
The concentrated semen had the supernatant removed and was extended again 1:23 with yolksugar-glycerol extender, which was composed of 10 parts of egg-yolk, 5.5 parts of glycerol and 84.5 parts of sugar media (glucose 1.8 gm, lactose 4.4 gm, raffinose 5.4 gm in 100 ml).
The extended semen was cooled to 5°C at semi-rapid rate by keeping it in small test tubes which were kept standing directly in 5°C cold air room. The 0.2ml of the cooled semen was placed directly on a block of dry ice after 23 hours of glycerol equilibration.
Semen frozen in pellet form on dry ice was transfered into plastic tubes (diameter 1.2cm, height 5.0 cm) which were made to stand in holes made in dry ice. Then the plastic tubes were sealed with plastic-aluminum film using the electric heat sealer. The small plastic tubes were stored in liquid nitrogen until use.
The frozen semen was carried to the mares owned by farmers in crushed dry ice by A. I. technicians.
Inseminations were carried out immediately after thawing, in which the 520 pellets of semen were added to 20ml of the thawing media in ampoules kept at 3035°C.
The thawing media were a mixture of following solutions and were stored at 4°C or at room temperature after heating repeatedly in ampoules. 1) Skim milk 100ml 2) Buffer solution 77.0ml. Composed of; Sod. citrate 1.57gm, Sod. carbonate 0.075gm, Pot. carbonate 0.085 gm, Glucose 2.7 gm, P-aminomethylbenzensulfonamide 0.2gm, Distilled water up to 100ml. 3) Glycerol 3.0ml. The pH of the mixed media…7.0 (adjusted by 5.5% citric acid solution).
The mares were inseminated 13 times per heat under conditions of the routine work and diagnoses to determine conceptions were performed by the rectal palpation. Inseminations to 93 mares, each of which was inseminated only during one heat, resulted in 43 conceptions. Fertility was 46.7 per cent, except one mare for which we could not know the result (Table 1).
Semen conditions in successful inseminations were as follows ;
Storage period. … 1110 days, Survival rates of sperm.…1565%, No. of pellets inseminated …
3-11, No. of sperms inseminated.…63-31.24 (×10⁸⁾).
Relationships of no. of spermatozoa inseminated and conceptions were as follows; 34 (×⁸⁾)… 50% (2/4), 56…50% (5/10), 7-8…76.9% (10/13), 9-10…50% (1/2), 13-14…42.8% (3/7).
Although the number of mares was too small and further observations should be needed, these results suggest that inseminations with 78 (×⁸⁾) spermatozoa would be enough to get high fertility when mare heat condition and insemination technique are good.

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A total of 71 primiparous, lactating rats were devided into 9 groups, with regard to the litter size and the duration of lactation. The number of suckling was adjusted to 4, 8 or 12 on the next day after parturition and the young were allowed to suckle for 7, 14 or 21 days, respectively. After weaning, each mother rat was placed with a fertile male and the intervals from weaning to the first post-weaning estrus, mating and conception were examined by daily inspection of vaginal smear. Results obtained are as follows:
1) The interval between weaning and the first post-weaning estrus (vaginal cornification) became shorter with increasing period of latctation. Multiple regression of the interval (Y) on the duration of lactation (X₁) and on the litter size (X₂) was Y=3.56-0.142(X₁-15.0)+0.050 (X₂-8.04), where b₁=-0.142 was highly significant, while b₂=0.050 was not significant.
2) The percentage of females mated up to the third post-weaning estrus was lower in 4-young nursing group, in comparison with 8 or 12-young nursing group. There was not significant difference between the percentage of females mated at the first post-weaning estrus and that of the second estrus.
3) Out of 55 mated females, 51 were conceived. Number of corpora lutea, implantation sites or living fetuses examined at the 19th day of gestation was not different significantly among groups.

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The immunity of bull semen was examined. Rabbits were immunized with bull semen or bull seminal plasma with Freund's complete adjuvant to take the anti-bull-semen serum or the anti-bull-seminal plasma serum.
The precipitation reactions wih these two sera resulted in that "the forms of the plates of precipitations" showed both a rectangel shape. In double diffusion method in agar plate (Ouchterlony method), the anti-bull-semen serum was recognized to have more than two antigens and the anti-bull-seminal plasma serum to have mere than four against both semen and seminal plasma. No difference was found among semens taken from three different bulls and anti-bull-semen serum concerned with the appearance of the precipitation lines in Ouchterlony method. No line was detected between anti-bull-seminal-plasma serum and swine seminal plasma.

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As a kind of protective agent against freezing-damage of animal cells, dimethyl sulfoxide (DMSO) was compared with glycerol concerning boar sperm. The semen was collected by means of artificial vagina from the 3 healthy boars. The basic diluent was composed of the 20% egg yolk SGS solution (IIDA& ADACHI: 1966).
At the first trial, seminal plasma was replaced by the 1st solution, with no protective agent included, using a centrifugator at a room temperature of 25°C. The material was cooled from the room temperature to 10°C at the rate of 1°/16 min. At 10°C, this material was added with the same volume of 2nd solution which contained various percentages of protective agent. After equilibration at 10°C for 2 hours, the material was frozen to -79°C at the rates of 1°/min from 10°C to -20°C and of 5°C/min from -20°C to the final temperature.
The results are summarized as follows:
1) The evaluation of toxic effect of DMSO (5, 7, 10 and 20% v/v) at the stage of pre-freezing was found to be equal to the toxic effect of 7% glycerol at 10°C within 6 hours.
2) In the observation of freeze-thawed sperm motilities, 1, 5, 10 and 20 % DMSO showed some protection of sperm cells against freezing damage, however they were less effective than that of 7% glycerol level.
3) Within those concentrations of DMSO used for this experiment, 5% level was the most effective for sperm motility.
At the second trial, the 2nd solution (2, 5, 10 and 15% DMSO and 14% glycerol) was added as described above but at 5°C, and then the material was frozen in pellet form (NAGASE & NIWA : 1964) at once.
4) From the above experimental result it was revealed that the order of effective concentration of DMSO was 5, 2.5, 1 and 7.5% in the final levels, however, there was no levels that were superior to 7% glycerol.
5) There are no significant diflerences analized respectively between the control and 5% and 2.5% DMSO levels, and 7.5% level showed highly significant difference between the other levels (Fig. 2).
From these results, the available percentage of DMSO on freezing of boar sperm was approximately 5%.

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