[in Japanese]

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Experiments were carried out to arrest rheotaxis-like orientation of dead spermatozoa in fine tubes by increasing viscosity of the diluents, and then to observe the inherent rheotaxis of live spermatozoa by using these highly viscous diluents.
Semen was diluted either with Tyrode's solution (rabbit) or with Ringer's solution (cock). The viscosity of the diluents was increased by adding 5% gelatin, and 7% glycerol was supplemented to prevent the freezing damage. The fine tubes of 250350μ in diameter were primarily used in this experiment. Tubes of 3 mm in diameter or rectangular cells about 0.15 mm depth were also used for the comparison when the semens were diluted with the highly viscous solutions. The stream of semen diluted with the highly viscous solution was stopped by pouring either dry ice-alcohol (fine tubes) or ice water saturated with NaCl (3 mm tubes). Then the former was defreezed at 4°C and the latter was sliced after fixing in cold 10% formalin before the observation of the directions of spermatozoa under microscope in the coagulated state.
Results obtained are summarized as follows:
1. When the rabbit semen diluted with the Tyrode's solution were allowed to flow in the fine tube at streaming velocity of 100 μ/sec. and 50 μ/sec., dead spermatozoa showed positive rheotaxis-like orientation but negative at 25 μ/sec. If the semen was diluted with the gelatin solution, the rates of spermatozoa pointing upstream, downstream and transverse direction approached to 25, 25 and 50% respectively in both fine tube and 3 mm tube.
2. The live spermatozoa of rabbits which were diluted in the gelatin solutin pointed upstream, downstream and transverse directions in a proportion of 1 :1 :1 respectively at the streaming velocity of 100 p/sec. in the fine tube. This proportion was not different among upper, middle and bottom layers in the tube. In the 3 mm tube, their percentages approached to similar proportions as in the case of dead spermatozoa.
3. When the cock semens diluted with the Ringer's solution with or without gelatin were allowed to flow in the fine tubes at the streaming velocities of 50, 25 or 12.5 μ/sec., the proportions of spermatozoa toward transvers direction were 60-70% in all cases regardless of the conditions employed in this experiment, and the proportions of upstream ones and downstream ones changed by the streaming velocities. In the rectangular cell, these three directions were 25, 25 and 50% respectively. There were few differences between live and dead spermatozoa.
From these results it was suggested that there were no rheotaxis inherent in rabbit and cock spermatozoa.

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Data on sex ratio in swine were collected from the experiment stations of animal husbandry in Akita(Ak), Gumma(Gu), Kanagawa(Kn), Chiba(Ch), and Fukuoka(Fo) Prefectures. They covered five breeds, Large White (W), Yorkshire (Y), Landrace (L), Hampshire (H), and Duroc(D), and their hybrids (Hyb). They were collected froma total of 12, 528 which consisted of 2, 739 in Ak (W, Y, L, H, Hyb), 1, 362 in Gu (W, L, H, D, Hyb), 5, 936 in Kn (W, Y, L, H, D, Hyb), 1, 958 in Ch (W, Y, L, H, D, Hyb), and 533 in Fo (W, L, H, Hyb). The results obtained from each breed were discarded and only the results obtained from all the breeds as a whole were described in the present report. These results were compared with those given in the preceding parts of the present series of studies.2-7) Analysis on sex ratio was performed by the same method as mentioned in part 1 of this series.2) The classes which showed a shift of sex ratio to either sex were cited mainly in tables.
The results obtained from this study are summarized as follows.
A shift to male (high sex ratio) in the total sex ratio was observed only in Kn. The lowet sex ratio (not significant) was noted in Gu. There were, however, no significant differences in sex ratio among the five stations (Table 1). There were boars and sows showing a shift of sex ratio in their offspring in all the five stations. The percentage of these boars and sows was in the same range as presented in previous reports^2-6) (Table 2). A shift of sex ratio was seen in one or two classes of litter series in all the stations, except that in Kn. A high sex ratio was seen in these stations, ex-cept one class in Ch (Table 3). In one or two classes of litter size, a shift of sex ratio (a low sex ratio in allthe stations, except one) was recognized in all the stations, except that in Ak (Table 4).
There was no seasonal shift of sex ratio at all in any station. Significant differences among the seasons, however, were seen only in the station of Gu (Table5). A shift of sex ratio was found in neither natural mating nor artificial insemination. There was no significant difference in sex ratio between the two typesof mating (Ak Table 6). A shift of sex ratio was noted in one or two classes of paternal age in three stations (Ak, Gu, and Ch). It was high in these stations, except one. A high sex ratio was seen in one class of maternal age bothin Kn and Fo (Table 7). A shift of sex ratio was observed in one or two classes of age difference between the parents in all the prefectures, ex-cept Ak. There were shifts of sex ratio both to the same and to the opposite sex of the older parent (Table 8). Shifts of sex ratio (both low and high) were observed in two classes of maternal age at the first mating and of calendar year in Ch, respectively (Tables 9 and 10).

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The estrogen levels in the peripheral blood serum of 5 mares (2 Percherons and 3 Bretons) were estimated by a radioimmunoassay throughout each gestation period. Blood samples were collected from each jugular vein at intervals of 2 to 3 days from Day 0 (the day of the last breeding) to Day 45 of the gestation, then during the rest gestation period, samples were taken at intervals of 15 to 30 days. Each serum sample (0.1 to 1.0 ml) was extracted by ethyl ether, and the dried extract was applied to a microcolumn of Sephadex LH-20 with the solvent system of benzene and methanol, 85:15. The eluate after being added with each of the tritiated estrogens, was combined with each specific antiserum and assayed. This radioimmunoassay method, originated by MAKINO¹²⁾ had sufficient accuracy and reliability for these estimations. The recovery rate showed, 80100% (for E₁), 7085% (for E₂) and 5075% (for E₃), and the water blank showed, 04 pg, 08 pg, and 03 pg, respectively. The working range shown in the standard curve corresponded with 0 to 500 pg for each estrogen.
The result obtained was summarized as follows:
1) The serum concentration of E₁ remained below 40 pg/ml from Day 0 to Day 75, then increased to 190.5 (158.7222.2) pg/ml at Day 120 and sharply up to the average value of 3682.2 (2582.05744.0) pg/ml at Day 210, and the high levels continued for about a month. It then declined sharply towards 107.9 pg/ml at Day 300, with a fluctuation at around Day 255 to 270.
2) The E₂ concentration showed a similar pattern as like as that of E₁: it remained below 15.2 pg/ml from Day 0 to Day 75, 46.5 (30.162.9) pg/m_l at Day 120, then began to increase rather sharply up to Day 210 (1080.4: 496.51826.5 pg/ml) and the average highest value obtained was 1740.6 pg/ml (at Day 240). From the Day around 210, the average E₂ concentration remained high for about 2 months, then it declined sharply to 71.3 pg/ml at Day 285.
3) The E₃ level showed large variations within and between individuals, and no collelation was observed throughout the gestation.

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A radioimmunoassay (RIA) for simultaneous measurement of 4-androstenedione (A), testosterone (T), and 5α-dihydrotestosterone (DHT) in 1 ml of canine plasma was demonstrated. The plasma was extracted by methylenedichloride, then was separated to the proper 3 fractions by Sephadex-LH-20 microcolumn (solvent system: cyclohexane 85/benzene 15/methanol 5). The individual steroid in each fraction was assayed by the RIA. In these assays, an anti-A-3-BSA rabbit serum was used for plasma A, and anti-T-3-BSA serum for both of plasma T and DHT. Each assay for these 3 plasma steroids showed to be satisfactorily sensitive, specific, and accurate. To obtain the basic 3 steroid levels in both of peripheral and spermatic vein plasma, each 8:00 AM sample was collected from 15 male dogs. The plasma steroid levels thus obtained were (ng/ml and SD); in peripheral vein, (A) 0.86±0.75, (T) 1.19±1.05, (DHT) 0.30±0.25, and in spermatic vein, (A) 30.2±18.5, (T) 157±98, (DHT) 17.5±10.7.
Analysing these data, a significant correlation between the two vein plasma levels was suggested. In order to estimate the normal, or common diurnal variations of these plasma steroid levels in male dogs, 5 animals were employed, and each peripheral plasma sample was collected every an hour or two, throughout each 24 hours period. In these data, frequent intermittent peaks of plasma steroids were observed in each dog, and these were thought their own diurnal changes.
It was concluded so, the peripheral plasma levels of A, T and DHT were correlated to those in spermatic vein, and these steroid levels was seemingly reflected by the episodic secretions from the gonad.

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The activity of glucose-6-phosphate dehydrogenase (G-6-PDH) in hamster and pig eggs was his-tochemically examined by the Rudolph and Klein method. Hamster eggs were collected under follow-ing conditions: 2 hrs after ovulation for unfertilized eggs, 4 hrs for penetrated eggs, 11 hrs for pro-nuclear eggs, 32 hrs for 2-cell eggs, 52 hrs for 4-cell eggs, 65 hrs for 8-cell eggs, and 76 hrs for blastocysts. Pig eggs were collected on Day 2 and Day 13 (2 and 13 full days after mating).
As for hamsters, a strong activity of G-6-PDH was observed in 1-cell eggs, and diformazan gra-nules produced by this enzyme were spread evenly throughout the cytoplasm of unfertilized and pene-trated eggs, while in pronuclear eggs such were especially abundant in the perinuclear region. In the eggs at 2-cell stage, the G-6-PDH activity grew to be weak, and the intensity was maintained as late as blastocyst stage. In cleaved eggs, the diformazan granules were located in the perinuclear region and the periphery of the cytoplasm, though in blastocysts the granules became uniformly spread in the cytoplasm of both inner cells and trophoblasts.
In uncleaved pig eggs on Day 2, a strong activity of G-6-PDH was observed, and the diformazan granules were located among lipid droplets which were closely packed in the cytoplasm. In elongated blastocysts on Day 13, a weak activity was observed, and the diformazan granules were uniformly spread in the cytoplasm of trophoblasts. Some difference in the amount of diformazan granules was observed among trophoblasts.
The results obtained from this investigation and former studies concerning steroid dehydrogenases seem to suggest that hamster and pig eggs at preimplantation stages utilize NADPH produced by G-6-PDH for biosynthesis of steroids.

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