1) Observations were made on the growth of rabbits only fed the green wild white clover. From the results of chemical analysis, the authors have already reported that the contents of moisture, crude fiber and ash of this clover are significantly changeable through the year, showing the seasonal variation. The contents of ash and crude fiber are to be examined successively in detail. From the results obtained in this experiment, it is shown that young litter rabbits were raised on the feed consisting of only the green wild white clover and including no water nor common salt for seven months after weaning and that they have reached almost the standard level of growth during the period.
2) Moreover, it is outstanding to find that no abnormal growth has been apparently observed in the young rabbits during the whole experimental period, although common salt is said to be essential to the herbivorous animal.
Perhaps the reason is as follows: As already repo-rted in the previous paper (KAMETAKA, M. and S. HARUYAMA J. Agr. Chem. Soc. of Japan, 28:309, 1954.) the content of sodium is more than that of potassium in the wild white clover, judging from the ratio of equivalent, and a certain balance is maintained between the content of sodium and of potassium in the body of rabbits even though no common salt is given to the growing rabbits as source of supply of sodium.

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1) In this experiment the digestibility of the wild white clover was determined by using the usual collection method at three different stages of growth in rabbits fed in the same manner as emp-loyed in the first experiment (No. 1 of this series of reports).
2) The three stages of growth were as follows the Ist stage when body weight reached 0.4 to 0.5 kg in May, 1948, the 2nd stage when body weight reached 1.9 to 2.0 kg in September, 1948, and the 3rd stage when body weight reached 2.4 to 2.5 kg in June, 1950.
The results indicate that there was no difference in the digestibility of all components between the lst and 3rd stage, but, at the, 2nd stage, the diges-tibility of dry and organic matter was a little lower.It may be thought that this fact is due to the dep-ression of the digestibility of crude protein and crude fiber, and, moreover, that of nitrogen-free extract.
As already reported in Parts 1 and 5 of the series of our studies (Kametaka, M. and S. Haruyama :Jap. J. Zootech. Sci., 23, 140, 1953, and Kametaka, M. and K. Hatakeyama: J. Agr. Chem. Soc. of Japan, 28, 391, 1954.), the content of crude fiber in the wild white clover increases significantly at a time immediately after the fall of blossoms and the contents of lignin and cutin are apt to increase at that time.
Accordingly, the fall in digestibility at the 2nd stage seems to be attributed to the result mentioned above, that is to say, the deterioration of nutritive quality of the wild white clover. But, from the re-sults of chemical analyses of the wild white clover fed in these digestion trials, it is hardly recognizable that the content of crude fiber increases immediately after the fall of blossoms.
Consequently, it may be necessary to take into consideration the living condition of the rabbits, especially the activity of microorganisms in the digestive tract at that period, as well as those results described in Parts 1 and 4 of the series of our studies in performing experiments of this kind.
3) The average digestibility of chemical compo-nents in the wild white clover was as follows : dry matter 87, organic matter 87, crude protein 91, true protein 84, crude fat 64, crude fiber 78 and nitrogen-free extract 88.

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A great advance has been made in the knowledge of the carotenoid metabolism in animal body, especially the absorption mechanism in the digestive organ, including the site and manner of the conversion of β-carotene into vitamin A. The rapid progress in the technique of chromatographic separation of various carotenoid compounds is a good help to treat the problem of carotenoid metabolism in animal body. Still there remains a large undecided aspect of carotenoid metabolism in laying hens, although excellent pioneer works of Palmer and his school have provided us a splendid knowledge of this field.
Recently in Japan, some poultry farmers have adopted the high green forage feeding method for laying hens for two reasons: one is to raise the atchability of egg and the other to economize feeding costs. As no data were hitherto found in the nutritional study on high green forage feeding in poultry, a study on carotenoid metabolism was conducted at first. The results of the study are summarized as follows:
1. When a large quantity of green herbage (115-170g a day) was given to laying hens, the content of xanthophylls in the egg yolk was increased rapidly to a great extent (6-8 mg of xanthopylls in 100g of egg yolk). But the rate of transfer of the feed xanthopylls to egg yolk was reduced, only 2-5% of this pigment appearing in the egg yolk.
2. In such case, the vitamin A content of egg was also large, indicating 200-480 βg in 100g of egg yolk. Calculation was made to find the efficiency of provitamin A of feed, and about 5-10% of provitamin A were converted into vitamin A of egg yolk. From these data, it has been proved that the hen ossesses a strong potency to produce vitamin A in egg from provitamin A with a higher efficiency than the rate of transfer of xanthophyll in egg.
3. Generally, the hen is considered to belong to the xanthophyll animal and its absoption ability of β-carotene is weeker than that of xanthophylls. This is true when we consider the xanthopyll-carotene ratio of the blood, ovary, skin, fat and other tissues of the hen. But from the result of feeding with dosage of green herbage containing abundant β-carotene, the utilization of β-carotene by the laying hen does not seem to be slow and unefficient.
4. Some investigators claim that a large amount of xanthophyll hinders the absorption of β-carotene in the bird, but the authors are in the opinion that little fear exists in this respect if high green forage feeding is carried out with rational supply of concentrates.
5. From the results mentioned above, the high green forage feeding method is not objectionable in poultry husbandry.

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As silkworm feces contein a large quantity of carotenoid pigments, they can be used in poultry feeding as a pigmentation agent for egg yolk and also as an effective source of vitamin A for eggs.A study was conducted to know in what degree the carotenoid pigments of silkworm feces can be utilized for egg production and how much vitamin A will be formed in eggs from these pigments. Twobreeds of laying hens were used in this experiment, that is, White eghorn and Nagoya. Both breeds showed the average rate of egg production of 70 percent. The results are summarized as follows.
1. When silkworm feces were given to laying hens, in addition to the ordinary concentrates in which yellow corn was replaced by white corn, the egg color began to increase on the third day after dosage and this color change was completed in the following week. Examination of the egg yolk thus changed indicated a marked, less conspicuous and little increase in the amounts of xanthophyll, β-carotene and vitamin A, respectively.
2. The degree of change in the color of egg yolk depends on the dose of silkworm feces. The addition of 5 percent of silkworm feces to the poultry feed doubled the original xanthophyll content of the egg yolk, but that of more than 5 percent of silkworm feces resulted in showing the law of diminishing return. As for the influence on the β-carotene content of the egg yolk, the 5 percent addition of silkworm feces to the feed caused a smaller (only 50%) increase in this carotenoid of the egg than an increase in xanthophyll. An additional dosage of more than 5 percent of silkworm feces resulted in the similar tendency of the law of diminishing return in the β-carotene content of the egg, as shown in the xanthophyll content. On the other hand, the 5 percent addition of silkworm feces to the feed showed almost no response in the vitamin A content of the egg yolk. Even in the case of a 30 percent addition of silkworm feces to the feed caused almost no change in the vitamin A content of the egg yolk in all the four experimental birds except one which showed only 20 percent increase of the vitamin A content of the egg yolk in this case.
3. Sudden stop of dosage of silkworm feces was followed by the decrease in the xanthophyll content of the egg whick started on the third day after the stoppage and which continued for the next week. The β-carotene content of the egg also decreased in the same way, but rather to a smaller extent than did the xanthophyll content. On the contrary, the vitamin A content of the egg yolk did not show any decrease for three weeks after the cessation of carotene supply.
4. The rate of transfer of carotenoids from silkworm feces to the egg was calculated as well as the rate of conversion of biologically effective caro tenes of silkworm feces into vitamin A of the egg. The data are indicated in table 9.
5. Although silkworm feces are an excellent feed supplying carotenoid pigment for poultry, as shown in the above experiment, the use of this stuff in more than 5 percent of poultry feed is not profitable for laying purposes, because the silkworm feces contain an unknown substance which causes diarrhea in birds in the case of eecess dosage and induce the reduction of egg production and vitamin A formation. For the purpose of pigmentation, an addition of 1 to 3 percent of thisstuff to poultry feed is recommendable.

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Although many experiments on metabolism of the chick embryo have been reported, most of them were conducted by using dissected eggs in different stages of growth. It would be very convenient for observation of the genesis of chick embryo if the existence and growth of the embryo could be maintained inside the egg shell opened partly or without wholeshell.
In this regard the author prepared an 'open-windowed egg' and kept it alive in an incubation bottle. The chick embryo was found to keep its growth and genesis for a week or so under certain conditions of temperature, humidity and gas ventilation in a onestoried incubation bottle. Moreover, a twe-storied incubator was devised, the upper space of which was in contact with the contents of an incubating egg and the lower space of which with the shell part of such egg. In this incubation bottle the genesis of an egg could be maintained for 20 days by supplying oxygen gas under appropriate conditions.
Many experiments were performed with this incubator and good results obtained by supplying oxygen with 90-100% humidity to the upper space(in contact with the contents of the egg) and oxygen with 60-75% humidity to the lower space(in contact with the shell part).

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Many studies have been conducted on pathogenic bacteria of the intestines of animals, but very few on non-pathogenic bacteria. However, non-pathogenic bacteria have a close relation with the health of animals. Recently it has been disclosed that the non-pathogenic bacteria have a great inf-luence on health and nutrition of animals by producin many sorts of vitamins, exercising an action of digestion on indigestible feed, etc. Consequently, it is important to know the regular bacteria existing in the intestines of animals.
Several reports have been published on some species of the bacteria existing in the human inte-stines, but very few studies have been made on the same subject in animals. Particularly, no systematic studies seem to have been performed, by using herbivorous, carnivorous and omnivorous animals comparatively, or using animals of the same species, to find out any difference in the bacterial flora due to different sorts of feed.
The author has carried out many experiments to study herbivorous animals, including an Indian elephant (Elephas planifrons FALCONER & CAUTL-EY), black rhinoceros (Rhinoceros bicornis L.), Japanese deer (Cervus nippon nippon TEMMINCR), Grant zebra (Equus guagga grante DE WWINTON), dromedary camel (Camelus dromeddarius L.), etc., carnivorous ones, including striped hyena (Hyaena crocuta ERXLEBEN), racoon-like dog (Nyctereutes procyonoides viverrinus TEMMINOK & SCaLEGEL), lion (Felis Leo L.), etc., omnivorous ones, including a chimpanzee (Pan satyrus L.), Japanese black bear (Ursus thibetanus japonicus CHLEGEL), mantled baboon (Papio hamadryas L.), pigtailed monkey (Macacar nernestrina L.), etc., and moreover, domestic animals and fowls, including a goat, sheep, cow, horse, pig, rabbit and chickens. Particularly, in chickens, the author examined the influence of feed, especially the quantity and qua-lity of protein contained in it, on the intestinal bacteria.
In the experiments mentioned above, plate Cul-tures were usually used. As they have many defects in counting bacteria and selecting a certain species of bacteria, the author adopted in this study the dilution method with a selected medium. As the selected mediun, the E. C. and M. E. medium was used for the cultivatian of bacteria of Escherichis coli type, the Koser's medium for the Aerobacter group, the same medium including "konnyaku" flakes for the onnyaku mannan-decomposing Aero-dacter group, the S. F. medium for Enterococci, the A. D. medium for Streptococi, and bouillon with liver slices and litmus milk for the Clostridium welchii group.
Experiments were performed on chickens with the feed containing 20% or 40% protein from different source, such as fish meal, soy-bean oil meal and milk casein.
Part I of this report deals with the organisms of Aerobacter, Escherichiia, Streptococci and Clost-ridium welchii detected in experiments on the istribution and frequency of appearance bacteria existing normally in the intestines of animals. The results obtained concerning the appearance of each of the groups are as follows.
It has been said that few organisms of Aero-bacter appear in the animal intestines, but in this study a good many of them appeared in most omni-vorous, some herbivorous and a few carnivorous animals. In the case of chickens, the appearance became less frequent according to an increase in protein content of their feed.
The Konnyaku-mannan decomposing Aerobacter group appeared in the herbivores in a large number in the omnivores in a small number, and in the carnivores in a much smaller number.
The frequency of appearance of the Escherichia coli type was particularly low in cattle. It was lower in the carnivore than in the herbivore. Among domestic animals, it was low in cattle, horses, sheep, goats, and rabbits, but high in swine.
In chickens it was as high as in swine, but no difference was observed in the frequency when they were given different sorts of feed.

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The present study was conducted to clarify the histological differentiation of the adrenal cortex of the bovine fetus and to determine the time when the sudanophilic lipid appears for the first time in the adrenal gland. Twenty bovine fetuses, from 13 to 43cm in C.R.L., collected at a slaughter house in Osaka were used in this study. Fixed with 10% formalin, one of the adrenals of each fetus was paraffin-sectioned at 7μ in thickness and stained by several methods; the other was frozen-secti ned at 25μ and stained with Sudan IV-hematoxylin. The observations are summarized as follows.
At the stage of a 13-cm C. R. L., the cortical parenchyma was composed of irregular cell cords alone. At that of a 16-cm C.R.L., beneath the capsule were revealed rather slightly smalloepitheloid cells grouped in spherical clusters. A layer of these clusters of cells is to be termed zona glom-erulosa. By the stage of a 24-cm C. R. L., the spherical clusters beneath the capsule developed further and expanded horizontally along the capsule with widening connective tissue spaces among them. The cells of z. glomerulosa were smaller and stained darker than those of z. fasciculata. Histolo-gical differentiation of the adrenal cortex of the bovine fetus seemed to be almost completed essen-tially by the stage of a 30-cm C. R. L., because little changes were found in the gland after this stage.
Few sudanophilic lipid droplets were found in the parenchyma of the adrenal cortex of a 19-cm-long fetus. In a 24-cm-long fetus, however, sudanophilic droplets were contained in some cortical rPlls, especially in the glomerular and the inner fasciculate cells in considerable abundance. From the stageof a 27-cm C. R. L., on all adrenals contained lipid droplets in the cortex with no exception. From the stage of a 27-cm C.R.L. on, lipid droplets gradually increased their size and number in the glomerular cells.
As the lipid contained in the adrenocortical cellsis said to have an intimate relatiorship with hormone production, it may be concluded presumably from the above observations that the adrenal of the bovine fetus may shows in some manner the hormone-formation or the secretory activity, at least after the growth stage of a 27-cm C. R. L.

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Broody hens of Nagoya breed were subjected to lighting by electric lamps, and the effect on broodiness and non-productive period after the cessation of broodiness was investigated. The following time conditions of lighting were used : for 20 hours from noon to 8:00 A. M. in experiment A, for 13 hours from 5:00 P. M. to 6:00 A. M. in B, and for 7 hours from 5:00 P. M. to midnight in C. The results are as follows:
1. The light treatment was effective for interrupting broodiness, which was observed after 2.68 days in experiment A, 3.27 days in B and 4.45 days in C, respectively, on the average.
2. There was no relationship between the broody period prior to the treatment and that under the treatment.
3. Non-productive periods after the cessation of broodiness averaged 9.94 days in A, 7.75 days in B and 5.10 days in C. The difference in days between experiments A and B was not significant at the 5% level.
4. There was no relationship between the nonproductive period and the total broody period or the broody period under the treatment.

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S. C. White Leghorn chickens of both sexes, including castrated wales, were employed at six weeks' intervals during the growth period to determine the rate of calcification of the stearum and tibia. They were killed at 1, 42, 84, 126 and 168 days of age, the calcium content and dried matter content of the sternum and of the tibia analyzed, and the decrease rates of the cartilage of the sternum measured. In addition, the indices of bone length to body weight were calculated.
The results are as follows.
1. In a statistical study on a relationship between the calcium content and the age, the calcium content (in%) of the chicken sternum and the age extending from 42 to 168 days showed a relationship expressed in a straight line. As for the tibia, the linear regression was allowed in the same limited range of the age by setling a log scale.
Significant differences were found among the slopes of regression lines obtained from the sternia of chickens of both sexes, including castrated males, the sharpest slope being shown, in the female, and the gentlest in the castrated male. In the tibiae, no significant difference was found between the corresponding slopes of the normal and the castrated male, but the female showed a significant, sharpe slope.
2. With regard to the dried matter contents of the sternia and the tibtae, it was found that differences among the three sexual groups were similar to those in the calcium content among the same groups.
3. In the indices of bone length to body weight, no significant differences were found among the female, the normal and the castrated male groups.
5. The cartilages of the sternal crest and metasternum showed a decrease with the growth of the chicken. The decrease of the cartilage area of the sternum was most rapid in the female and more rapid in the normal male than in the castrated male.

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Herring oil was added to the basal chicken feed consisting of corn, rye, rice-bran meal and soybean cake and its effect on egg production was investigated. Addition of 1 or 2g of herring oil to 100g of the basal ration was most effective and gave better results in autumn than in spring.

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The CO-derivative of myoglobin obtained by the method described in the previous report and that of haemoglobin were observed spectrophotometrically.
Light absorption spectra of these two typical haemoprotains showed no difference in various species of animals.
However, there was a considerable difference between light absorption spectra of CO-myoglobin and those of CO-hemoglobin, since in the former the maximum was recorded at 577, 540∼541 mμand the minimum at 560 mμ, while in the latter the maximum was recorded at 570, 539∼540 mμand the minimum at 555 mμ.
A nomogram was made (Fig. 4) by means of this difference between CO-myoglobin and CO-haemoglobin, and by using this nomogram, myoglobin contents of muscles of different species of amounts of total red pigments in the meat were determined.
It has been proved that the amount of total red pigments in the meat decreases in horse meat, beef mutton and pork in this order and that a considerable difference exists among different muscles of the same individual.
The myoglobin content of the red muscle pigment increases with the increase of total pigments, in other words, the deeper the red color of meat, the more myoglobin is contained in the meat.

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For the purpose of detecting bovine mastitis by catalase activity, the catalase activity of colostrum and normal milk was determined gasometrically by measuring the volume of oxygen set free with Warburg's manometer. Moreover, we conducted experiments not only on the relationship between the activity and the leucocyte count and pH, but also on the influence of the storage temperature and period of time on the activity. The main results obtained are as follows:
1. Colostrum shows higher activity than normal milk. Though the average activity is 4271.6 μl per 1.5cc one day after calving and decreases re markably with the lapse of time, it does not reduce to the level of normal milk even one week after calving.
2. The catalase activity of the milk, giving a negative Hotis test, from cows with no history of mastitis is 71.4μl. That of the milk, giving a positive Hotis test, from the similar cows, is 165.5μl. That of the milk from cows which have history of mastitis but are producing apparently normal milk is 541.3μl. Therefore, a catalase activity of 1651μl or higher would indicate a suspicion of subclinical mastitis, so that the activity as high as 540μl would especially be a strong indication.
3. In normal milk, a greater part(about 2/3) of the catalase activity is caused by bacteria, whereas in mastitis-infected milk a greater percentage(about 80%) of the activity is caused by leucocytes.
4. In experiments where milk was stored at 10°, 20° and 30°C for 21/2 and 5 hours, respectively, no difference in activity was noticed among them.

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1. By using the mechanical separation method, which originated from OHIBNALL's idea in the preparation of leaf proteins, the tissue of fresh forage grass was divided into three main parts, i. e., a fibrous, a concentrated and a water-soluble part. In this method, fresh grass was ground at first by the Waring blendor and the fibrous part was separated from a thick green juice after filtration through a cloth filter. Then the green juice was centrifuged and a green precipitate (the concentrated part) consisting mainly of chloroplastic fragments, was separated from almost transparent liquid (the water soluble part). Each of the three portions was evaporated to powder in vacuum at low temperature for the purpose of analysis.
2. Two kinds of forage grass were used in the experiment: Kentucky 31 fescue and white clover. Preliminary analysis was carried out to determine the general chemical composition and plant pigments content of these two grasses.
3. The distribution of dry matter of grass in the three main parts differed largely between fescue and clover. Fescue transferred much dry matter to the fibrous part, and white clover did the same to the water-soluble part.
4. The bistribution of nitrogenous compounds of grass in the three main parts was alike in fescue and clover. Both grasses transferred about half of the nitrogenous compounds to the concentrated part which contained originally more than 62 percrude of crude protein in its dry matter. This content of cent protein is much higher than the protein content of spinach chloroplast determined by COMAR
5. The distribution of crude fat of grass differed somewhat between fescue and clover. Fescue transferred half of the crude fat to the concentrated part, while white clover transferred almost equal amounts to the three parts. This fact suggests the different physical structure of fatty substance between fescue and clover.
6. The distribution of crude ash of grass in the three parts was alike in fescue and clover, indicating an interesting fact. About 70 to 78 percent of the total ash of grass were transferred to the watersoluble part. The detailed analysis of this water soluble ash (along with insoluble ash) is now in progress.
7. More than 50 percent (up to 60 percent) of the plant pigments (chlorophyll, xanthophyll, β-carotene and cryptoxanthin) of fresh grass were transferred to the concentrated part of the plant tissue, regardless of the grass breed. But some difference was found between fescue and clover, a larger amount of chlorophyll ressidue being shown in the fibrous part of clover.
8. The composition of the three parts of the forage grasses used in this experiment is shown in detail in table 21.

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