With Four Graphs.) PREVIOUS experiments (l, 2, 3) have shown that pullets display an increasing retention of calcium and phosphorus during the 2 to 3 weeks before laying begins. In these experiments laying was associated with an increase in phosphorus excretion, and with a lowered calcium-phosphorus ratio in the excrement. It has been pointed out (3) that this extra phosphorus excretion occurs discontinuously and appears to be related to the period of shell formation.The experiments described in the present communication were carried out in order to trace the metabolism of sodium and potassium as well as of calcium and phosphorus in the non-laying and laying pullet. At the same time the effect of sodium chloride on the retention of calcium was studied.PART I. SCHEME OF EXPERIMENT.

SINGULARLY few records of mineral balance studies on poultry have appeared in the literature. The difficulty of separating faecal excrement from urinary excrement has probably been responsible for this. At the same time such studies may yield information, even if the separation of faeces and urine is not carried out.Hainan (i) conducted balance experiments on two pullets over a period of 14 weeks; he found that storage of calcium, phosphorus and nitrogen occurs during only a short period before laying; the extra demands of the bird for these constituents during egg laying is met by increased retention from the food. He further concluded that an excessive calcium intake may lead to a depletion of phosphorus reserves, and he considered egg production to be associated with increased phosphorus catabolism, the phosphorus lost in this way exceeding that voided in the eggs.Russell and McDonald (2) carried out somewhat similar experiments on the calcium and phosphorus balance of pullets receiving either calcium carbonate or calcium citrate. Eight pullets were placed on a weekly balance for 5 weeks. The authors concluded that'' negative or very slightly positive balances and a lowered excretion of calcium in the droppings during egg production are excellent evidence that the laying pullet can utilise calcium from calcium citrate as well as from the carbonate for egg production." The balances, in other words, did not indicate any difference in the utilisation of calcium from either source. Russell and McDonald also noted that "in general during egg production more phosphorus is excreted in the droppings than during non-production, and more than is necessary to form tricalcium phosphate with the excretory calcium."The experiments described below were begun in 1929 with a view to securing information on the changes in calcium and phosphorus metabolism which accompany the onset of egg production.

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Staining for ester-linked protein phosphorus (ELPP) gave positive reactions with both of the main lipoprotein zones (P-1 and P-2) of paper electropherograms of egg yolk in certain buffers, including veronal buffer, pH 8.6. Treatment of the yolk with 1.0 M NaCl or with complexing agents (ethylenediaminetetraacetate, veronal–citrate buffer) resulted in the appearance of larger amounts of ELPP in the phosvitin (PT) zone. The distribution of the livetin zones suggested that the lipoprotein zone P-1 overlapped the greater part of the γ-livetin zone. This suggestion was supported by the results of immunoelectrophoretic experiments.Yolks in which the phosphorus had been labelled with P32 were separated by ultracentrifugation into (a) granule material; (b) low density fraction (LDF); and (c) the soluble livetin fraction. Paper electrophoretic studies of whole egg yolk and of these preparations showed (a) that the P-1 zone of electropherograms of whole egg yolk in veronal buffer comprised all of the LDF plus most of the γ-livetin plus a proportion of the phosvitin that depended on the strength of the salt solution used to disperse the yolk, the nature of the buffer, and the absence or presence of complexing agents; and (b) that the P-2 zone comprised the lipovitellin fraction together with more or less of the phosvitin. Dispersion of the yolk in 1.0 M NaCl, pretreatment of the yolk with ethylenediaminetetraacetate, or use of veronal–citrate buffer led to the appearance of relatively high proportions of the total protein-bound phosphorus and P32 in the 'fast' phosvitin (PT) zone.

An application of radioimmunoassay to measurement of concentrations of estradiol and estrone in peripheral plasma of hens is described. Evidence is presented that the estradiol measured by the procedure was essentially estradiol-17β.For 13 ovulatory cycles the average concentration of estradiol was 123 pg/ml with a range from 36 pg/ml to 284 pg/ml. The corresponding average concentration for estrone was 64 pg/ml with a range from 14 pg/ml to 138 pg/ml. For 12 days of no ovulation the average concentration of estradiol was 142 pg/ml with a range from 42 pg/ml to 364 pg/ml. The corresponding average value for estrone was 91 pg/ml with a range from 18 pg/ml to 242 pg/ml.The results are interpreted as demonstrating the existence of peak levels of plasma estradiol at 22–18 h and again at 6–2 h before ovulation, and a minimal level at 14–10 h before ovulation. The corresponding plasma estrone concentrations displayed a similar cycle but the earlier peak and the minimum occurred 2–4 h later than the corresponding values for estradiol. Such peak values were not demonstrable on days when ovulation did not occur.The results are discussed briefly in relation to variation in the levels of blood progesterone during the ovulatory cycle.