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During the passage of the egg down the oviduct, the egg shell membrane has essentially three sizes of particles on the outer surface. Membranes from the mid-isthmus have small surface grains, visible only with staining, that range in size from one to ten microns. They occur at approximately 270 per mm.2 increasing to approximately 2700 grains per mm.2 in the posterior isthmus. These grains give a positive reaction for glyoxalbis-(o-hydroxyanil) and a negative reaction for carbonate. The grains are acid soluble and seem loosely attached since they seem to agglomerate during staining. They diminish in number when the egg has progressed into the tubular shell gland and are rarely found once the egg has reached the shell gland. Possibly the grains act as nuclei for the depositions of the secretions of the tubular shell gland. The particles on the egg shell membrane in the tubular shell gland range from 28 to 90 micronsin in diameter. These are the mammillae which are enmeshed in the fibers of the outer shell membrane. They give a positive reaction for carbonate. The mammillae on membranes from the shell gland range from 60 to 150 microns in diameter. They stain uniformly with nine different histochemical stains, showing a relatively even chemical composition. They grow in size during deposition of shell mineral and eventually touch each other, forming small irregular gaps which become part of the pores of the finished shell.

SINCE the work of Benesch et al. (1944) and Gutowska and Mitchell (1945) it has been generally assumed that the enzyme carbonic anhydrase plays an important role in egg shell formation by supplying the carbonate anion of calcium carbonate through the reaction CO2 + H2O⇄ H2CO3. This assumption is based on (a) the significantly greater carbonic anhydrase activity of uterine epithelium as compared with other oviduct tissue (Common, 1941), and (b) the reduction of shell deposition after feeding sulfanilamide, described by Benesch et al. (1944) and Gutowska and Mitchell (1945). Sulfanilamide and other unsubstituted sulfonamides, such as acetazolamide, are carbonic anhydrase inhibitors. As such they interfere with the reabsorption of bicarbonate in the kidney and, through the concomitant metabolic acidosis, often cause diuresis (Burger, 1960). It is therefor possible that sulfanilamide and other carbonic anhydrase inhibitors interfere with shell deposition by causing diuresis and not by limiting the supply of …

Bone cell activity and the composition of the femur of laying hens were studied during 7 days of calcium depletion on a 0.13% calcium diet and 7 days of calcium repletion on a 3.2% calcium diet. Histologically, only cortical bone showed clear signs of bone resorption and osteoclastic activity during the depletion period. The number of osteoclasts in medullary bone varied little from control values throughout both calcium depletion and repletion, except for a significant increase on the first day of depletion. The major histologicalchange in medullary bone was a marked increase in the number of osteoblasts on the third, fifth and, to a lesser extent, seventh, day of depletion. The number of osteoblasts in medullary bone was positively correlated with its osteoid content and negatively correlated with its degree of calcification. Alkaline phosphatase activity of medullary bone increased with the time the hens had been on the calcium-deficient diet. Returning the hens to the 3.2% calcium ration caused, within one day, a significant increase in medullary bone calcification, a decrease of osteoblast numbers to, or below, control levels, and a drastic reduction in alkaline phosphatase activity of medullary bone. The significance of these findings in relation to the control of bone cell populations and the functions of medullary bone is discussed.

SOME of the processes which accompany the thinning of the thick albumen during egg storage are loss of CO2, loss of water, and water movement from the albumen to the yolk. Numerous experiments on storage of eggs at high CO2 concentrations as well as experiments on different methods of shell sealing have demonstrated that the CO2 concentration within the egg affects the rate of albumen and yolk quality breakdown. Funk (1944), Korslund et al. (1956) and Mueller (1958) reported that relative humidity during storage, which controls the rate of evaporation, has little or no effect on albumen and yolk deterioration. Less is known on the relationship between albumen quality breakdown and water movement from albumen to yolk. Heiman and Wilhelm (1937) found no significant correlation between yolk index (ratio of yolk height to yolk width) and percentage firm white or albumen index for eggs kept for the same length of …

1. 1. The normal buffer line of oxygenated blood from laying hens is: [HCO3−] = 76·162−7·055 pH, that of deoxygenated blood [HCO3−] = 98·763−9·614 pH. 2. 2. The amount of base released from hemoglobin during deoxygenation (ΔX) decreases from 0·62 m-equiv. base/m-equiv. O2Hb at pH 7·24 to 0·46 at pH 7·75. 3. 3. The difference in slope between the two buffer lines is due, at least in part, to the influence of pH and pCO2 on ΔX. 4. 4. Avian blood seems to have a lower buffer value than mammalian blood. This is compensated, at least partially, by a greater release of base during deoxygenation. 5. 5. Egg shell formation causes an increase in the nonvolatile acid content of blood of 3·36 m-equiv/l.

IT HAS been reported by Atwood and Weakley (1924) as well as by Smith (1931) that infertile chicken eggs lose CO2 during storage. According to Smith (1931) the CO2 loss of individual eggs may begin at a rate of 9.0 mg. per day and gradually drop to 0.1–0.2 mg. per day, at which level it may continue for 100 days or more. Smith (1931) also found that the rate of CO2 loss depends on temperature. The CO2 content of fresh eggs has been determined by Mathieu and Urbain (1908) and Straub and Donck (1934). These investigators found that the albumen of fresh eggs contains approximately 55 mg. CO2 and that there is no CO2 in the yolk. Numerous experiments on storage of eggs at high CO2 concentrations as well as experiments on different methods of shell sealing have demonstrated the importance of CO2 with regard to maintenance of interior . . .

The plasma calcium and phosphate response to iv injection of PTH was studied in laying hens fed diets containing 2.26% or 5.00% calcium, both during periods of egg shell formation and when no egg was in the shell gland. Compared with results for mammals, the response was considerably larger, more rapid and more transient. With PTH injection at 0 min, plasma calcium (5.00% calcium diet only) and phosphate were significantly decreased at 5 min. This was followed by an increase in the concentration of both ions until a maximum was reached between 20 and 30 min. After 60 min, plasma levels of pullets fed the 2.26% calcium diet were again practically normal. The maximum plasma calcium and phosphate response for the 5.00% calcium diet was more than twice as large as for the 2.26% calcium diet, while the stage in the egg cycle had no effect on the response curves. During the period of hypercalcemia the increases in plasma calcium and phosphate were closely correlated (r = 0.733, p < 0.001), suggesting that the r...

1. 1. During shell formation the organs of the laying hen received the following percentages of the cardiac output: kidneys (arterial), 9·96; shell gland, 8·58; liver (arterial), 7·05; heart, 5·78; magnum, 5·70; duodenum, 4·81; femora, 4·08; spleen, 3·37; tibiae, 2·62; isthmus, 1·06; pancreas, 0·70; colon, 0·40; metatarsi, 0·10; cerebellum, 0·09; parathyroids, 0·06; thyroids, 0·04; adrenals, 0·03. 2. 2. The Evans Blue method gave consistently higher estimates of cardiac output (277 ± 16 ml/kg per min) than the indocyanine green method (177 ± 11 ml/kg per min). 3. 3. On a weight basis, the parathyroids received the highest blood flow (13·5 ml/g per min), possibly because they may include the carotid body. Blood flow rates to the duodenum and bones were higher than in mammals.

IN AN earlier paper (Mueller, 1962) it was reported that the diuretics mercuhydrin, cardalin and acetazolamide interfered with egg shell formation. The present report deals with the aldosterone antagonist spironolactone which causes diuresis in mammals. Reviews on aldosterone antagonists have been prepared by Bartter (1960) and DeStevens (1963). It is generally agreed that spironolactone causes diuresis by blocking the urinary electrolyte effects of aldosterone and other mineralocorticoids. Thus, spironolactone causes an increase in sodium and a decrease in potassium excretion. All evidence indicates that spironolactone acts at the kidney level; it does not inhibit aldosterone formation at normal dosages. The effectiveness of spironolactone in mammals is limited. The changes in electrolyte balance are usually observed only in the presence of hyperaldosteronism (Salassa et al., 1958) or in adrenalectomized animals (Kagawa, 1960). In intact rats and dogs Kagawa (1960) found no changes in urine volume and sodium excretion after spironolactone doses …

INVESTIGATIONS of the seasonal variation of egg production in different geographic locations (for reviews see Huston et al., 1957; Ragab and Assem, 1953) have led to the conclusion that high environmental temperature is one of the major causes of the decline in egg production during summer. In most of these studies it was difficult to separate the effect of age from the effect of the seasonally changing environment. The results of experiments with layers of the same age are less conclusive. Warren et al. (1950) found little difference in annual egg production between an uncontrolled environment, where the mean daily temperatures ranged from about 30°F. in winter to about 85°F. in summer, and a controlled environment where a constant temperature of 65°F. was maintained. Huston et al. (1957) reported that White Leghorn hens laid as well at a constant temperature of 90°F. as hens kept in an environment where mean…

PREVIOUS reports on the vitelline membrane have been conflicting with respect to its chemical composition, physical structure and average thickness. Liebermann (1888) reported that the vitelline membrane consisted of keratinous material. Lacaillon (1910) found three layers, the middle layer being cellular in structure, whereas the external layers were fibrous. Moran and Hale (1936) also reported three layers and concluded that the two external layers consisted of mucin while the middle layer was keratinous. McNally (1943) found that the vitelline membrane of the freshly ovulated yolk was formed from the collagenous membrane which lines the inner surface of the follicular epithelium. He reported that this membrane became swollen and was covered with a a layer of mucin during its passage down the oviduct. Reports on the average thickness of the vitelline membrane range from 15 microns (Lacaillon, 1910) to 47.74 microns (McNally, 1943). The mechanisms involved in the development of yolk …

AN EARLIER paper (Mueller, 1961) dealt with the performance of pullets at constant temperatures of 32°C. (90°F.), 13°C. (55°F.) and in an environment where temperature cycled from 32°C. to 13°C. and back to 32°C. every 24 hours. Using performance at a constant temperature of 13°C. as a base, continuous exposure to 32°C. reduced egg production of survivors 28%, average egg weight 15% and feed intake 32%. Little is known about the mechanisms which cause these adverse effects of high temperature. One possibility, suggested by further analysis of the data reported in 1961, was environmentally induced nutritional deficiencies. Hill and Dansky (1950) reported that, while daily nutrient requirements appeared to be relatively constant absolute quantities, requirements expressed as percentage of the ration were related to feed intake. Since the rations used from 1958 to 1960 (Mueller, 1961) were formulated in terms of required nutrient levels per unit weight of feed, the …

IN RECENT years an increasing number of poultrymen have been keeping layers for a second year of production. While adequate information is available on changes in egg quality during the pullet year, experimental evidence on the effect of age on egg quality during the second year of production is, with the exception of egg weight, relatively limited. Romanoff and Romanoff (1949) reviewed the literature on the changes in egg weight as the age of layers increases. They state that in spring hatched pullets egg weight increases from October to February or March, then declines somewhat until midsummer, but subsequently increases until it approximates that of two year old hens in October or November. According to these authors two year old hens lay their largest eggs in December or January and their smallest eggs in May, June and July. Benjamin (1920) in an experiment with Single Comb White Leghorns, found no …

BENNION and Warren (1933) and Warren and Schnepel (1940) reported that shell thickness is reduced markedly if hens are subjected to high environmental temperatures. Their experiments further suggested that the depressing effect of high temperature is accentuated by high humidity. The mechanism or mechanisms which cause poor shell quality at high air temperatures are still not definitely established. Conrad (1939) found that blood calcium levels were reduced at high environmental temperatures. From this evidence Warren and Schnepel (1940) concluded that: “The decrease in shell thickness when the birds are subjected to high temperatures is probably due to a reduction in calcium content of the blood.” Warren and Schnepel (1940) also reported that hens kept at 95°F. consumed about 26% less feed than hens kept at 60°F. It is possible that this reduction of feed intake is at least partially responsible for the poor shell quality at high air temperatures. Finally, …

THE prevalence of egg quality problems as well as an expanded use of high energy diets for layers has increased interest in the effect of nutrition upon egg quality. It is well known that yolk color depends on the pigment content of the diet and that inadequate mineral and vitamin nutrition results in a high percentage of eggs with defective shells. With these exceptions, however, it has been difficult to find any definite relationship between nutrition and egg quality characteristics. This is especially true for albumen quality. Thompson et al. (1932), Card and Sloan (1934), Lee et al. (1944), and Griminger and Scott (1954) found that the energy content of the diet as well as the kind of cereals used caused no or only small differences in albumen quality. Despite the experimental evidence cited many poultrymen still maintain that high energy diets have a depressing effect upon interior egg quality …

The regulation of the appetite for calcium was studied in broiler chickens which had been trained to meet their calcium requirement by consuming a calcium supplement (10% Ca) provided separately from a calcium-deficient feed (0.12% Ca). Calcium supplement intake was reduced within 150 minutes after intravenous injection of 60 U/kg parathyroid hormone or continuous infusion of 0.077 mg/minute calcium into one common carotid artery. Supplement consumption was also depressed during the first 8 days of continuous infusion of 1 U/kg/hour parathyroid hormone; after 8 days the birds became refractory. Continuous infusion of 0.2 Medical Research Council (MRC) units/kg salmon calcitonin for 10 days or daily intramuscular injections of 1 mg/kg testosterone propionate and 2 mg/kg estradiol dipropionate for 12 days, either alone or in combination, had no effect on calcium appetite. It is concluded that the calcium appetite may be inhibited by increased concentrations of ionic calcium in blood and that the change in behavior is sufficiently rapid to play a role in the calcium homeostasis of birds.

A limited restraint harness was developed which permits long-term infusion of chickens under near normal conditions. The harness allows the birds to stand, sit, move forward and backward, and turn 180° in either direction without twisting the cannula.

Labeled microspheres were used to measure blood flow to the leg bones of the laying hen at 0, 3, and 30 min after iv injection of parathyroid hormone (PTH) (Wilson) or the synthetic 1 to 34 fragment of bovine parathyroid hormone (PTH 1-34). At 3 min, which corresponds to the hypocalcemic phase of the PTH response, blood flow to the combined femur, tibia, and metatarus was significantly reduced by PTH (Wilson) relative to 0 time and to carrier-injected controls. At 30 min, i.e., the time of maximum hypercalcemia in the hen, blood flow to these bones was significantly increased relative to 0 time. The results obtained with PTH 1-34 were similar, except that the decrease at 3 min was only significant in comparison with the controls injected with inactivated hormone. Femoral blood flow and the venous minus arterial calcium gradients across the femur were positively correlated, irrespective of sampling time (0 or 30 min) or type of injection (PTH [Wilson] or carrier). Taken together, these results suggest that there is a relationship between calcium mobilization from bone and the rate of osseous blood flow. Other organs which showed significant changes in blood flow after PTH (Wilson) were the adrenals, thyroids, and shell gland; the cerebellum, parathyroids, heart, spleen, liver, pancreas, duodenum, magnum, isthmus, and kidneys were not affected.

Autoradiography with tritiated acetazolamide indicates that osteoclasts of the hen and chick contain concentrations of carbonic anhydrase which are similar to those in pancreatic acinar cells. Grain counts of osteoblasts and osteocytes were not different from background. Thus, a sufficient quantity of carbonic anhydrase seems to be present in osteoclasts to be of physiological importance in bone resorption.

Carbonic anhydrase was localized at the light microscope level using labeled inhibitor autoradiography. Laying hens, 10-day-old chicks and adult female Japanese quail received injections of 1-25 µC 3H-acetazolamide/g body weight. After 3 hr (quail) and 24 hr (hen and chick), 2-mm3 pieces of tissue were removed under anesthesia, frozen at –160°C, freeze-dried, fixed with OsO4 vapor, embedded in Epon, sectioned at 1 µ and covered with NTB-2 emulsion. The following results validate the method: (a) the concentration of acetazolamide was 100 times greater in red blood cells than in plasma 24 hr after injection in the hen, indicating that free and loosely bound acetazolamide was rapidly cleared from the body; (b) no significant amounts of label were found in muscle, cartilage, adipose tissue and nuclei, which have no carbonic anhydrase activity; (c) except for the pancreas, there was good agreement among the grain counts for the different classes of birds; and (d) the autoradiographic localizations were in general agreement with concepts of tissue function. Carbonic anhydrase was localized in: the proximal, distal, collecting tubules and the mesangium of the kidney; the acinar, but not the centroacinar, cells of the pancreas; the tubular glands and the columnar cells of the shell gland; the lining cells, but not the zymogenic cells, of the proventriculus; erythrocytes; erythroblasts; heterophils; and lymphoid nodules, but not in fat and interstitial tissue of bone marrow.