[in Japanese]

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Two holstein cows were overfed with high quality grass silage and with supplemental rations consisting of a small amount of hay and some concentrates for a four month period which began at the time of the first estrus post-partum. The amount of the daily supply of grass silage was equivalent to about 7 percent of the body weight of the animals. Blood specimens were taken from these cows about every 3 days during the second estrous cycle. And the levels of free estrogen and progesterone in the blood plasma were bio-assayed respectively by Sulman's and by Hooker-Forbes' methods.
During the estrous cycle the level of blood estrogen showed a pattern with two definite peaks, one at the estrus and the other at the functional luteal stages, while the level of progesterone showed a pattern with one definite peak at the 9-12 th day of the luteal stage.
These patterns were similar to those of normal cows. Moreover, the cyclic changes of the genital organs seemed to be normal.

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It is very important to control bacterial contamination of bull semen not only for maintaning good fertility in artificial insemination by the semen but for preventing genital infection in the cow served by it. For this purpose sulfanilamide, penicillin and dihydrostreptomycin are customarily added to semen diluents. Since 1957 I have been studied on the prevention of bacterial contamination of bull semen.
The purpose of the work presented here was to investigate the actual condition of bacterial contamination of original bull semen. A total of 221 semen samples collected from breeding bulls were used as meterial. The results obtained were as follows;
1) Degree of the bacterial contamination of original bull semen was considerably high, the number of bacteria recovered being from 2×10³/ml to 1.7×10⁷/ml. In 48 (21.7 %) of the 221 semen samples examined, the number of bacteria was over 10⁶/ml (Table 1).
2) The number of bacteria in semen from the bull in which preputial douche had been performed before semen collection decreased markedly as compared to that without preputial douch (Table 1 and Fig. 1). The effect of the preputial duoche for preventing bacterial contamination of semen was confirmed experimentally (Table 2).
3) From 219 original semen samples 750 strains of bacteria were recovered as shown in Table 3. Almost all of them were determined as non-pathogenic or saprophytic bacteria. It was supposed that the bacteria were derived primarily from the preputial cavity of the bull.

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The purpose of the present studies was to investigated the actual condition of bacterial contamination of bull semen which was diluted, treated with penicillin and dihydrostreptomycin and stored at low tempreature. A total of 527 semen samples stored in some AI centers was used as material. The results obtained were as follows.
1) In spite of the treatment of with antibiotics, the recovery rate of bacteria from the diluted bull semen was unexpectedly high and increased from 28. 6% in 1957 to 56. 2% in 1961 as shown in Table 1.
2) However the number of bacteria recovered from bull semen treated with antibiotics was much less than that treated with no antibiotics. The number of bacteria in the antibiotics treated semen was also decreased when the semen was collected from the bull in which preputial douche had been performed before semen collection as compared to that without preputial douche (Table 2).
3) From 125 semen samples treated with antibiotics 20 genera (species) of bacteria were recovered, the main ones being Achromobacter, Yeasts, Pseudomonas and Alcaligenes (Table 3). In addition to the 4 genera of bacteria, Escherichia coli and Proteus were also dominant in the semen samples treated with no antibiotics (Table 4). As showen in Table 5, the species of bacteria recovered from the semen was simplified by the treatment with antibiotics. Except Yeasts, these genera of bacteria were generally resistant to penicillin and dihydrostreptomycin, and about 80% of them were Gram negative.

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A phase contrast microscope is one of the most valuable instruments for observing the living cells, . and is often used for the evaluation of sperm viability and mortility. However, there has not yet been reported of any case of its application to the counting of abnormal spermatozoa, since they are in motion. The fixed, stained semen smears are generally in use for counting of abnormal spermatozoa. If the spermatozoal features of the fixed, unstained smears observed with the phase contrast microscope are reasonably similar to those of the fixed, stained smears observed with the ordinary microscope, there is a possibility of simplifying the technique for counting abnormal spermatozoa, because the process of staining can be omitted in the former.
This experiment was carried out to devise a simplified technique for counting abnormal spermatozoa with the phase contrast microscope.
All the samples were collected from the bulls, boars and rabbits by means of artificial vagina.
1. The features of spermatozoa which were fixed in Orth's fluid for two hours or longer, mounted. with distilled water and then observed with the phase contrast microscope are nearly the same to those of living spermatozoa. The phase plate of positive low low contrast is superior to those of positive low contrast, negative medium contrast and negative high contrast for the purpose of observing the spermatozoa, since it makes possible to get the features of spermatozoa as if the spermatozoa were stained.
2. In the fixed, unstained spermatozoa observed with the phase contrast microscope, both the parts and the abnormalities therein of spermatozoa are readily distinguishable. Accordingly, it is very easy to count and classify the abnormal spermatozoa, including the acrosome cap abnormalities. But, some problems are found therein. Firstly, the determination of the abnormal stainability of nucleus is almost impossible. Secondly, the accurate numbers of spermatozoa with cytoplasmic drop are uncountable because of the breakdown of the drop in some cases. However, the former is mostly accompanied with the deform of the nucleus and the latter is a common problem of the fixed smears. Therefore, these problems are considered negligible for the purpose of the counting and classification of abnormal spermatozoa.
From the findings mentioned in 1 and 2 above, it is concluded that the counting and classification of abnormal spermatozoa are possible with the phase contrast microscope if the semen smears are fixed in Orth's fluid for two hours or longer and mounted with distilled water.
3. In the spermatozoa of bulls, boars and rabbits, although their features resemble closely, there are still minor differences among them. When the spermatozoa are observed with a positive phase, in the rabbits, the edge of acrosome cap looks dark all the way, making U-shape, but in the bulls and boars, the darkness of the edge is confined to the front part.
In addition to the above fact, a slightly dark zone is observed along the frontal edge of equatorial segment in the rabbits. Such a structure cannot be found in the bulls and boars.

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Effects of glycerol and phosphoglycerol on the respiration and motility of spermatozoa have beee studied with different species of domestic animals. Bull, stallion, boar, ram, goat and rabbit semen were collected by means of artificial vagina. Respiration of washed spermatozoa was measured in Warburg manometer at 37°C in the presence of glycerol (0.01 M) and, α and β phosphoglycerol (0.01 or 0.02 M). After 3 hrs of the incubation, motility and glycerol consumption of spermatozoa were examined.
Glycerol increased the rate of oxygen uptake and motility of bull and ram spermatozoa. These effects were low in goat and boar spermatozoa, and almost nil in stallion and rabbit spermatozoa. Glycerol consumption by spermatozoa was higher in bull and ram than in goat, and little in boar.
Phosphoglycerol (α or/and β) increased the rate of oxygen uptake of spermatozoa in all the species examined. On the whole, phosphoglycerol more increased the sperm motility than did glycerol, although the effect of the former was obscure in stallion and rabbit.
The cause of the species differences in glycerol and phosphoglycerol utilization by the spermatozoa was discussed in relation to the pathway of glycerol metabolism and the enzymic activity in the cells.

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