Twelve Holstein calves were fed on whole milk, and were administered by acetic, propionic and butyric acid, individually and as equimolar mixture or as 6:3:1 mixture, through the rumen cannula twice a day from 3 to 13 weeks of age, to determine the effect of the intraruminal constitution of volatile fatty acids on the metabolic activity of the rumen epithelium. The calves were sacrificed at the end of 13 weeks of age. Epithelium from the anterior dorsal blind sac was separated from the muscle layer. Two g of the rumen epithelium was incubated at 38°C for 3 hours in KREBS-RINGER bicarbonate medium at pH 7.2 in a 95:5 atmosphere of oxygen and carbon dioxide. Two hundred μ moles of acetate, propionate, butyrate, and an equimolar mixture of these three acids were added to medium.
Butyrate was the best substrate to be metabolized by the rumen epithelium from the all calves used. The amount of each volatile fatty acid metabolized was, in descending order, butyrate, acetate and propionate in all trials. It seems that the utilization of volatile fatty acids by the rumen epithelium of the young calves increased in a situation of the preferential utilization of butyric acid, independently of the intra-ruminal constitution of volatile fatty acids.

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The production of volatile carbonyl compounds (VCC) from milk constituents by Brevibacterium linens was investigated.
Washed cells of B. linens were inoculated to single component solution (pH7.0) of casein, amino acid, milk fat or glucose, and incubated on a reciprocal shaker at 21°C for 24hrs. Identification of produced VCC was made by thin-layer chromatography and light absorption of their 2, 4-dinitrophenylhydrazone derivatives. The results obtained were summarized as follows:
1) Larger number of VCC were produced from cultivated casein solution, than from milk fat and glucose. They were formaldehyde, acetaldehyde, acetone, pentanone-2 and heptanone-2. These VCC were selectively produced from a single amino acid solution cultivated by the same strain. Especially, the formation of acetaldehyde from aspartic acid, arginine, and glutamic acid, and that of acetone from glutamic acid, leucine and aspartic acid were significant.
2) When milk fat was cultivated, acetone was produced. n-Butyric acid was found to be as an original volatile fatty acid of acetone, and β-ke tobutyric acid was presumed to be adirect precursor of acetone.
3) Three kinds of VCC, i.e., formaldehyde, acetaldehyde and acetone, were produced from cultivated glucose solution. Pyruvic acid was a sole acidic carbonyl compound produced in the same cultivated solution and seemed to be a direct precursor of acetone.

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Althougt the non-protein nitrogenous compounds (NPN-compds.) such as urea and ammonium salts are known to be utilized as protein substitutes in ruminants, many of quantitative aspects concerning their utilization have not been elucidated. It has become more important than ever to determine (a) the degree of NH₃ fixed by rumen microorganisms, (b) the quantitative role played by each of bacteria and protozoa, and (c) the relative significance of rumen microorganisms in the net utilization of NPN-compds. These problems have hardly been subjected to an in vivo experimental test, because of the complexity of dinamic states of various N-components in the rumen, as well as of the difficulty of definite seperation among such components. But as for the metabolic pathways of NPN in the rumen, many informations have been accumulated since a few decades, and some N-components including bacteria and protozoa can be drawn, if partially, without any appreciable contamination. Under these circumstances, with the use of NPN-compds. labeled with ¹⁵N, a new kinetic approach appears to become available to throw light upon the problems mentioned above.
In order to make it possible to deal mathematically with the transference of ¹⁵N in the rumen, it is necessary to establish the simplest scheme possible about it. In this sense the scheme established in this paper (Fig. 1) is deboid of the possible re-entrance of absorbed ¹⁵N into the rumen through such a pathway as so-called "recycling of urea". Furthermore, the appearance of ¹⁵N into the protozoa-N pool is defined to be conducted only by way of bacteria, and any other pathway, if possible, is negrected. For the mathematical treatment of this scheme, it must be premised that each compartment in Fig. 1 (A, B and P) is held in the steady state. Such a steady state is assumed to be held at least under a regular feeding condition. On this premise the equations (9) and (11) could be obtained to express the changes of ¹⁵N atom-% excess in bacteria and protozoa, respectively.
The equation (9) shows that the curve for ¹⁵N atom-% excess of bacteria-N is decomposed into two straight lines when they are plotted on the semi-log coordinate. Applying this information to the results of our former experiments1), in which ¹⁵N-ammoniumcitrate (exp. I) and ¹⁵Nurea (exp. II) were administered independently to a cow, the following values were obtained for three unknowns in equation (9): A0'=0.610 atom-% excess, k1=0.463h-1, and r=0.064h-1 in exp. II (Fig. 2), and A0'=0.640 atom-% excess, k1=0.451h-1, and r=0.063h-1 in exp. I (Fig. 3). k1 means the decreasing rate of NH₃-¹⁵N that is caused by being utilized by bacteria per an hour, and the values obtained here indicate that the velocity of the utilization of NH₃-N by rumen bacteria is 45-46% per an hour. On the other hand, the velocity of the ingestion of bacteria-N by protozoa seems to be less than 6% per an hour, for r(=k2+k3) means the decreasing rate of bacteria-¹⁵N that is caused by flowing out toward the omasum as well as being ingested by protozoa. These velocities, however, do not necessarily mean the degrees of N conversions, because turnover times of ¹⁵N in compartments A and B must also be taken into consideration.
In order to obtain further informations about quantitative problems, it is essential to know all of the k values in Fig. 1, and a new experiment is accordingly now in progress in our laboratory.

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In our previous report2), it was proposed that some quantitative informations concerning the transference of NH₃-N in the rumen can be known by the use of kinetic method after the administration of ¹⁵N labeled substances. In the present report, ¹⁵N-urea has been given to a cow for the purpose of the further confirmation of that possibility, and of finding out the degree of the utilization of urea-N by rumen microbes.
In the present experiment (exp. III), 49.9g of 5.786 atom-% excess ¹⁵N-urea (N: 41.3%) and 223.6g of polyethylenglycol-4, 000 (PEG) were administered orally at the same time to the animal weighing 591kg, which had been maintained on such a ration as shown in table 1, and samples of rumen liquor were taken through the fistula with the lapse of time after the administration. The method of collecting bacteria and protozoa purely from rumen liquor is shown in Fig. 1. In table 2, ¹⁵N atom-% excess of bacteria-N, of protozoa-N, and of NH₃-N, concentrations of NH₃-N and PEG in the rumen, and excess amounts of NH₃-¹⁵N per 100ml of rumen liquor are listed.
As shown in Fig. 1, semi-log plots of the concentrations of PEG in rumen liquor are nearly on a straight line except the one measured at once after the feeding. This means that the volume of liquid in the rumen was kept nearly constant. Furthermore, the amounts of proteinN per unit volume of rumen liquor were also found to be nearly constant from one feeding time to the next except immediately after the feeding (Table 3). These observations are considered to show that the compartments, A, B, and P in Fig. 4 might be held in the steady state enough to be able to deal with mathematically.
In the scheme shown as Fig. 4, k6 means the decreasing rate of NH₃-¹⁵N that is caused by flowing out toward the omasum, and it can be equal to that of PEG which is calculated as 0.124h-¹ from Fig. 2. The net decreasing rate K(=kl+k5+k6) of NH₃-¹⁵N in compartment A can be obtained as 0.864h-¹ from the linear part of Fig. 3, in which excess amounts of NH₃¹⁵N per 100ml of rumen liquor are semi-log plotted. The value of k1, which means the decreasing rate of NH3-15N through being utilized by bacteria, can be obtained as 0.463h-¹ from the analysis of the changes of 15N atom-% excess of bacteria-N (Fig. 5), as discussed in our previous report2)
On the basis of these k1, k6, and K values, the following speculation can be made as for the degree of the utilization of urea: Since the turnover time of NH₃-¹⁵N in the compartment A is 1/K hours, and the rate of utilization of NH₃-¹⁵N by bacteria is k1 per an hour, the percentage of NH₃-¹⁵N utilized by bacteria can be obtained from k1×1/K×100, and this value, 53.6%, is considered to mean the degree of urea-N utilized by rumen bacteria in the present experiment. The percentage of NH₃-¹⁵N flowing out toward the omasum without either being utilized by bacteria or being absorbed through the rumen wall, is obtained as 14.5% from the expression of kg×1/K×100. The rest, ca.30% of urea-N is considered to be absorbed as NH₃ through the rumen wall.
On establishing the scheme, the possible re-entrance of absorbed ¹⁵N into the rumen was neglected, but it has been observed in this experiment. With the existence of such a pathway, the degree of urea utilization by bacteria might be slightly more than 53.6%. When ¹⁵N atom-% excess of bacteria-N measured dung full experimental period are plotted on the semi-log coordinate (Fig. 6), the decreasing part of the curve is expressed as a broken line. This can not be explained by the scheme, but may possibly be attributed to the re-cycling of ¹⁵N.

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The formation of complex of κ-casein with β-lactoglobulin on heating was reported by ZITTLE et al.⁷⁾ and LONG et al.²⁾ SAWYER et al.⁴⁾ found that the heat-induced complex ofβ-lactoglobulin and κ-casein was prevented by addition of sulfhydryl (SH) masking agent, N-ethylmaleimide, and suggested the importance of the disulfide bond formation in the association of two proteins. HARTMAN and SWANSON¹⁾. by means of disc electrophoresis, have observed the complex of κ-caseinand β-lactoglobulin on heating.
As the casein fractions and whey proteins exist as mixtures or complex to make up protein system in milk, the changes of and between proteins on heating milk may be different and more complicated than those observed in the model system made of separated protein fractions.
In a previous paper⁵⁾, the effects of heat on αs-, κ-and β-casein fractions with respect to their changes in the solubility in the presence of Ca ion, the electrophoretic and sedimentation properties, the digestibility and liberation of nitrogen and phosphorus were described. In the present experiment, to examine the effect of heating on natural protein system in milk, whole, αs-and κ-casein fractions were prepared from heated milk and some of their properties were investigated.Whole, αs- and κ-casein fractions were prepared from heated (80°C, 30min) skimmilk, and some of their properties were investigated.
αs-Casein fraction from heated milk was less sensitive to Ca ion than that from unheated milk. κ-Casein fraction from heated milk did not possess the ability to stabilize αs-casein against Ca ion. The heated κ-casein fraction and Ic-casein fraction prepared from heated whole casein retained the stabilizing ability.
From these results the loss of stabilizing ability of κ-casein from heated milk was assumed not to result from the change of κ-casein itself, but to be due to the interaction of κ-casein with whey protein fractions. The electrophoretic analyses gave the results to support this assumption.
When milk was heated in the presence of thioglycollic acid, this interaction was prevented and κ-casein fraction which retained the stabilizing ability was obtained.

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